Candidates against infection

ABSTRACT

The present invention relates to the use of plasminogen/plasmin and its derivatives as agents for enhancing host defense against infection or other infectious diseases. The invention also relates to a method for screening of compounds which enhance host defense against infection by evaluating the host defense against bacterial arthritis and spontaneous otitis media in an animal model.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.12/439,517, filed Feb. 27, 2009, which is a U.S. National Phaseapplication of PCT/SE2007/050585, filed Aug. 28, 2007, which claimspriority to the U.S. Provisional Patent Application No. 60/823,665,filed Aug. 28, 2006, each of which is hereby incorporated by referencein the present disclosure in its entirety.

FIELD OF INVENTION

This invention relates to compounds and methods for prophylaxis,prevention, and/or treatment of infectious diseases and necroticconditions affecting the extracellular matrix, especially due tobacteria. In particular, the invention relates to such compounds andmethods which result in improved infection defense, better cleaning ofnecrotic tissue as well as creating a functional and aestheticallysatisfactory tissue remodeling. The invention also relates to animalmodels for studying bacterial infection and tissue necrosis, andscreening methods for identifying and evaluating drugs and for enhancingtreatment methods against bacterial infection and tissue necrosis.

BACKGROUND

Infectious diseases are caused by pathogens such as bacteria and virusesand eukaryotic organisms ranging from single-celled fungi and protozoa,through large complex metazoan such as parasitic worms. Pathogenicbacteria may contain virulence factors that mediate interactions withthe host, eliciting particular responses from the host cells thatpromote the replication and spread of the pathogen. Viruses rely onsubverting the machinery of the host cell to produce their proteins andto replicate their genomes. Pathogens often colonize the host byadhering to or invading the epithelial surfaces that are in directcontact with the environment. Viruses rely largely on receptor-mediatedendocytosis for host cell entry, while bacteria exploit cell adhesionand phagocytic pathways (1). Pathogenic fungi, protozoa and othereukaryotic parasites typically pass through several different formsduring the course of infection; the ability to switch among these formsis usually required for the parasites to be able to survive in a hostand cause disease. During the initial hours and days of host exposure toa new pathogen, the innate immune system is the first line of defenseagainst invading pathogens. However, the initiation of specific adaptiveimmune responses is also required. Innate immune responses rely on thebody's ability to recognize conserved features of pathogens that are notpresent in the uninfected host. These include many types of molecules onmicrobial surfaces and the double-stranded RNA of some viruses. Surfacemolecules of microorganisms also activate the complement system totarget these organisms for phagocytosis by macrophages and neutrophils,and to produce an inflammatory response.

Bacteria have developed different strategies to escape from phagocytes.For instance, they can inhibit chemotaxis and phagocytosis, kill orcolonize the phagocytes. The phagocytic cells use a combination ofdegrading enzymes, anti-microbial peptides and reactive oxygen speciesto kill the invading microorganisms (2). In addition, they releasesignaling molecules that trigger an inflammatory response and begin tomarshal the forces of the adaptive immune system. Bacteria, on the otherhand, have developed different strategies directed against the adaptiveimmune system such as molecular mimicry, suppression of antibodies,hiding inside cells, or release of antigen into the bloodstream (3).

Intracellular pathogens, including all viruses and many bacteria andprotozoa, replicate inside a host cell, which they invade by one of avariety of mechanisms. Viruses rely largely on receptor-mediatedendocytosis for host cell entry, while bacteria exploit cell adhesionand phagocytic pathways. Protozoa employ unique invasion strategies thatusually require significant metabolic expense. Once inside,intracellular pathogens seek out a niche that is favorable for theirreplication, frequently altering host cell membrane traffic andexploiting the cytoskeleton for intracellular movement.

Staphylococcus aureus is a microorganism frequently associated withbacterial arthritis, which results in synovial inflammation, cartilageand bone destruction, and eventually joint deformity. Various animalspecies including mammals, birds and reptiles have been observed todevelop spontaneous S. aureus arthritis and are therefore potentialmodels for the induction of the disease.

The plasminogen activator (PA) system is a general proteolytie systemthat has been suggested to play an important role in the development ofdifferent types of arthritis. Plasminogen can be activated to thebroad-spectrum protease plasmin by either of the two physiological PAs,tissue-type PA UFA)or urokinase-type PA (uPA).

Otitis media is defined as inflammatory conditions of the ear. Otitismedia is the most common childhood disease except for the common cold.The most important etiological factor related to otitis media isbacterial or viral infections of the upper respiratory tract. Otitismedia is generally benign and a self-limiting disease, but despite this,the prescription rate of antibiotics is high. In fact, effects ofantibiotics in curing otitis media lack evidence and so far surgicalintervention is the therapy of choice for the treatment of recurrentacute otitis media (AOM) and chronic otitis media or otitis media witheffusion (OME).

It is well known that the immediate colonization by the patient's normalskin flora (i.g. S. aureus and Streptococcus pyogenes) occurs followinginjury. Especially after the introduction of penicillin G in the early1950s, which resulted in the virtual elimination of Streptococcuspyogenes as a cause of infection in thermally injured patients, S.aureus became the principal etiological agent of wound infection.Therefore S. aureus is one of the most common bacterium species onopen-wound infection. Incisional wounds and burn wounds are the mostcommon wound types observed in clinical practice.

Antibiotics and other antimicrobial drugs have been widely used intreatment of infectious diseases since the World War II era. The successof antimicrobials against, disease-causing microbes is among modernmedicine's great achievements. However, many antimicrobials are not aseffective as they used to be due to the development of drug resistance.A key factor in the development of antibiotic resistance is the abilityof infectious organisms to adapt quickly to new environmentalconditions. Over time, some bacteria have developed ways to circumventthe effects of antibiotics. Widespread use of antibiotics is thought tohave spurred evolutionarily adaptations that enable bacteria to survivethese powerful drugs. Antimicrobial resistance provides a survivalbenefit to microbes and makes it harder to eliminate infections from thebody. Ultimately, the increasing difficulty in fighting off microbesleads to an increased risk of acquiring infections in a hospital orother setting. Diseases such as tuberculosis, gonorrhea, malaria, andchildhood ear infections are now more difficult to treat than they werejust a few years ago. Drug resistance is an especially difficult problemfor hospitals harboring critically ill patients who are less able tofight off infections without the help of antibiotics. Heavy use ofantibiotics in these patients selects for changes in bacteria that bringabout drug resistance. Unfortunately, this worsens the problem byproducing bacteria with a greater ability to survive even in thepresence of the strongest antibiotics. These even strongerdrug-resistant bacteria continue to prey on vulnerable hospitalpatients. Therefore, there is an increasing awareness that noveltherapeutical strategies are highly needed to improve the infectiondefense against infect ion.

Necrosis is the name given to unprogrammed or accidental death of cellsand living tissue. It is less orderly than apoptosis, which are part ofprogrammed cell death. In contrast to apoptosis, cleanup of cell debrisresulting from necrosis by phagocytes of the immune system is generallymore difficult, as the disorderly death generally does not send “eat-me”cell signals which tell nearby phagocytes to engulf the dying cell. Thislack of signaling makes it harder for the immune system to locate andrecycle dead cells which have died through necrosis than if the cell hadundergone apoptosis. The release of intracellular content after cellularmembrane damage is the cause of inflammation in necrosis.

There are many causes of necrosis including injury, infection, cancer,infarction, invenomation and inflammation. Severe damage to oneessential system in the cell leads to secondary damage to other systems,a so-called “cascade of effects”. Necrosis is caused by special enzymesthat are released by lysosomes which are capable of digesting cellcomponents or the entire cell itself. The injuries received by the cellmay compromise the lysosome membrane, or may set off art unorganizedchain reaction which causes the release in enzymes. Unlike in apoptosis,cells that die by necrosis may release harmful chemicals that damageother cells. Biopsy material necrosis is halted by fixation or freezing.

Currently there are four major therapeutical methods to cure necrosis.The first is surgical removal, which is the most rapid, and therefore isrecommended when large necrotic areas or thick eschar present. Thesecond is mechanical removal, which includes hydrotherapy. dextranomersand wound irrigation. The third is enzymatic removal 1, the enzyme usedis mainly collagenase (eg: Santyl), however, the effect is too slow wheninfection presents; and fourthly is through autolytic method, which isvia enzymes in wound fluid but the effect is extremely slow. However,none of the four treatment methods provide a functional andaesthetically satisfactory necrosis removal and tissue remodeling.Therefore, a novel therapeutic strategy is in great need in order toachieve a successful removal of necrosis.

Current therapeutic methods for treating infections such as bacterialarthritis, open wound infection, otitis media and necrosis havedrawbacks as discussed above. Therefore, there is a great need in theart for improved strategies for treating infections in general.

SUMMARY OF THE INVENTION

The present invention relates to the surprising discovery thatcomponents, of the plasminogen-activation pathway, and compounds withthe capacity to activate plasminogen can be used for new and improvedstrategies for infectious diseases and tissue necrosis. An aspect of theinvention relates to the ability of plasminogen, or other members of theplasminogen-activation pathway or compounds with the capacity toactivate plasminogen, to play a role in protecting against e.g. S.aureus-induced arthritis and open wound infection by activatinginflammatory cells, killing bacteria, removing necrotic tissue andenhancing cytokine expression. Such infection conditions further includeinfectious diseases and other diseases where tissue infection iscommonly observed, for instance during tissue-specific infectiondefense, overall-body infection defense, acute infections, chronicinfections, chronic ulcers, open wound infection and diabetic ulcers.

In certain embodiments, the invention relates to a method for theprophylaxis, prevention and/or treatment of infectious diseasecomprising administering an effective amount of a compound that is acomponent of the plasminogen-activating pathway or has the capacity toactivate plasminogen directly or via the plasminogen-activating pathway.

In certain embodiments, the compounds with the capacity to activateplasminogen directly or via the plasminogen-activating pathway includestreptokinase, saruplase, alteplase, reteplase, tenecteplase,anistreplase, monteplase, lanoteplase, pamiteplase, staphylokinase andrecombinant forms and variants of the components of theplasminogen-activating pathway.

In certain embodiments, the infectious disease is a bacterial infectiousdisease or a viral infectious disease.

In additional embodiments, the bacterial infectious disease is selectedfrom otitis media, bacterial arthritis, gingivitis, periodontitis,conjunctivitis, wound infection, surgical wound infections, necrosis,pneumonia, injuries in the respiratory organs caused by burns and/orinfections and infected chronic leg ulcers in patients suffering fromdiabetes, venous or combined venous/arterial insufficiency or infectiousarthritis or the like, injuries in the joint tissues caused byinfections, preferably otitis media or bacterial arthritis.

In certain embodiments, the composition comprises a combination of twoor more compounds. In certain embodiments, the composition furthercomprises at least one antibiotic agent.

In additional embodiments, the antibiotic agent is selected from thegroup consisting of tetracyclines, amphenicols, beta-lactams,penicillins, sulphonamides, maerolides, lincosamides, streptogamins,streptomycins, quinolones and metronidazoles.

In certain embodiments, the subject is mammal, and in particular ahuman. In additional embodiments the subject is deficient in plasmin orplasminogen. The deficiency can he congenital, acquired and/or local.

In certain embodiments, the compound is administered systemically,locally, topically, intravenously, intramuscularly, subcutaneously, viainhalation, intrathecally, via local injection, via intra-articularinjection or rectally. In a preferred embodiment topical administrationand/or local injection are used.

In certain embodiments, the compound is administered in combination witha suitable polypeptide carrier and/or one or more stabilizing agent

In yet additional embodiments, the compound is administered at, a doseof 0.0001 to about 1 g, preferably 0.005 mg to about 100 mg, morepreferably from about 0.05 to about 50 mg. The dose in mg is in relationto square centimeter of infected area (i.e. mg/square centimeterinfected area).

In further embodiments, the administration of the compound is repeatedat least once, preferably at least every day.

In further embodiments, the administration is performed by applying awound dressing, comprising the compound of the present invention, to aninfected area.

In additional embodiments, the invention relates to a method for theprophylaxis, prevention and/or treatment of infectious disease, whichcomprises administering a pharmaceutical composition comprising aneffective amount of a suitable compound to a subject in need of suchtreatment.

In certain embodiments, the invention relates to a pharmaceuticalcomposition for the prophylaxis, prevention and/or treatment ofinfectious disease comprising an effective amount of a suitablecompound.

In certain embodiments, the invention relates to a kit for use in theprophylaxis, prevention and/or treatment of infectious diseasecomprising an effective amount of a suitable compound and at least oneantibiotic or antimycotic agent, in separate vials.

In certain embodiments, the invention relates to a method of identifyingan agent that is useful in promoting host defense against infection,comprising the steps of: a) administering a test agent to an animalhaving a bacterial arthritis; b) evaluating at least one of theparameters: (i) the extent of killing bacteria, (ii) necrotic tissueformation, (iii) inflammatory cell activation, (iv) cytokine expressionof the infection e.g. bacterial arthritis: c) comparing the chosenparameter(s) of step (b) with a control value; and d) selecting any testagent for which the chosen parameter(s) is (are) more beneficialcompared to the control value as an agent useful in promoting hostdefense against infection.

In certain embodiments, the animal is selected from a member of thegroup consisting of a wild-type animal and a transgenic animal lackingendogenous expression of plasminogen.

In certain embodiments, the invention relates to a method for diagnosisof an ongoing infection, comprising determining the diagnostic presenceof plasminogen.

In certain embodiments, the invention relates to a kit for use in thedetermination of plasminogen in a sample from a patient, wherein thesample is from body fluids, serum, excreted waste products, such asurine or faeces, exhalation, air, or the like, in order to determine anongoing infection and/or the effect of an ongoing treatment, comprisinga plasminogen determinant and means for collecting, storing and/orexamining the patient sample.

DESCRIPTION OF THE INVENTION

The present results in the ear showed that plasminogen plays a role inprotecting against the spontaneous development of chronic otitis media.The present results also suggest using plasminogen for clinical therapyof certain types of otitis media. Therefore, these findings suggest thatcomponents of the plasminogen-activation pathway have a role inprevention and treatment of any infectious disease as a novelpro-inflammatory factor. In particular, the pro-inflammatory effects ofcomponents of the plasminogen-activation pathway include activatinginflammatory cells, killing bacteria, removing necrotic tissue andenhancing cytokine expression and improving proper tissue remodeling.This conclusion is drawn based on the understanding of the overall hostdefense mechanism against all the infectious pathogens, the vast andversatile infectious diseases induced by S. aureus, which is the majorbacterium species used in our studies, and various infection modelsstudied in this patent application (including infectious arthritis, burninduced infection, incision induced infection and otitis media).

Infectious diseases currently cause about a third of all human deaths inthe world, more than all forms of cancer combined. Many types ofpathogens cause disease in humans. The most familiar are viruses andbacteria. Other infectious pathogens are eukaryotic organisms, rangingfrom single-celled fungi and protozoa, through large complex metazoansuch as parasitic worms. Each individual pathogen causes disease indifferent, way, which makes it challenging to understand the basicbiology of infection. However, all pathogens share the ability tointeract with host cells in ways that promote replication and spread ofthe pathogen, but these host-pathogen interactions are diverse.Pathogens often colonize the host by adhering to or invading through theepithelial surfaces such as skin surface that is in direct contact withthe environment, Intracellular pathogens, including all viruses and manybacteria and protozoa, replicate inside a host cell, which they invadeby one of a variety of mechanisms. Viruses rely largely onreceptor-mediated endocytosis for host cell entry, while bacteriaexploit cell adhesion and phagocytic pathways. Protozoa employ uniqueinvasion strategies that usually require significant metabolic expense.Once inside, intracellular pathogens seek out a niche that is favorablefor their replication, frequently altering host cell membrane trafficand exploiting the cytoskeleton for intracellular movement. Besidesaltering the behavior of the individual host cells, pathogens frequentlyalter the behavior of the host organism in ways that favor spread to anew host. Pathogens evolve rapidly, so new infectious diseasesfrequently emerge, and old diseases acquire new ways to evade humanattempts at treatment, prevention and eradication. Furthermore, with thegreat progress against infectious diseases such as vaccines andantibiotics, pathogens have also developed drug resistance through 1)producing art enzyme that destroys the drug, 2) altering moleculartarget of the drug so that it is no longer sensitive the drug, or 3)preventing access to the target. Therefore, drug resistant pathogens area growing problem.

Despite the various ways that pathogens have developed to invade humanbeings, there are only limited patterns that host defense machineryreacts against the infection. The host defense machinery includes bothadaptive immune system and innate immune system. Whereas the adaptiveimmune system remembers previous encounters with specific pathogens anddestroys them with the help of the innate immune system when they attackagain, the innate immune system is not specific to a particular pathogenin the way that the adaptive immune system is. Thus, the innate immunesystem is the first line of defense against invading pathogens and it isalso required to initiate specific adaptive immune responses.

Innate immune responses rely on the body's ability to recognizeconserved features of pathogens that are not present in the uninfectedhost. These features include, for instance, peptidoglycan cell wall andflagella of bacteria, as well as lipopolysaccharide (LPS) onGram-negative bacteria and teichoic acids on Gram-positive bacteria,zymosan, glucan and chitin in the cell walls of fungi and thedouble-stranded RNA of most viruses. Many of these pathogen-specificmolecules are recognized by Toll-like receptor proteins on inflammatorycells. In vertebrates, microbial surface molecules also activatecomplement, a group of blood proteins that act together to disrupt themembrane of the microorganism, to target microorganisms for phagocytosisby macrophages and neutrophils, and to produce art inflammatoryresponses. The phagocytic cells use a combination of degralativeenzymes, antimicrobial peptides, and reactive oxygen species to kill theinvading microorganisms. The inflammatory cells also degrade thenecrotic tissue formed in consequence of the infection through secretaryor internal enzymes. In addition, they release signaling molecules thattrigger and inflammatory response and begin to marshal the forces of theadaptive immune system. Cells infected with viruses are recognized bymacrophages through dead/dying cells, Toll-like recptors and defensins.These macrophages further respond by secreting inflammatory cytokines,hydrolyzing viral proteins in phagolysosomes and present the viralproteins at the nearby lymph nodes and spleen to activate moreinflammatory cells. The complement system can also recognize theviruses, activate the inflammatory cells to kill the virus and furtherinduce adaptive immune system to generate antibodies.

As discussed above, in the innate immune system, the inflammatory cells(neutrophils and macrophages) play the central role in the host defenseagainst all kinds of infection, ranging from viruses and bacteria,single-celled fungi and protozoa, to large complex metazoan such asparasitic worms. The inflammatory cells actively seek, engulf anddestroy pathogens directly or through a variety of cell-surfacereceptors such as Toll-like receptors and complement receptors. If apathogen is too large such as large parasites, a group of macrophagesand neutrophils will gather around the invader. Activated macrophagesalso recruit additional phagocytic cells to sites of infection.Inflammatory cells also secrete a variety of signaling molecules tomediate and amplify the inflammatory response. In the B cell-mediatedadaptive immune response against infectious pathogens, newly generatedantibody binds to the antigens on the pathogens through its Fab fragmentand to the surface receptors (FcR) on the inflammatory cells (mainlymacrophages) through its Fc fragment and therefore link the inflammatorycells to pathogens and further kill them.

Based on the disclosed discovery, the administration of plasminogen andits derivatives plays a pluripotent role in protecting against e.g. S.aureus-induced bacterial arthritis, open wound infection and otitismedia by activating inflammatory cells, killing bacteria, removingnecrotic tissue, enhancing cytokine expression and promoting normaltissue remodeling. All these effects are different aspects of the potentfunctions that inflammatory cells exert against all kinds of infections.A working hypothesis has been developed in order to explain all the datawe have. In this hypothesis, the key point is that plasminogenpotentiates the activity of inflammatory cells and therefore mediatesthe processes such as killing bacteria, removing necrotic tissue,enhancing cytokine expression and promoting normal tissue remodeling. Asindicated above, the inflammatory cells (neutrophils and macrophages)play the central role in the host defense against all kinds ofinfection, ranging from viruses and bacteria, single-celled fungi andprotozoa, to large complex metazoan such as parasitic worms. Therefore,discoveries reported in the current invention support the conclusionthat plasminogen and its derivatives are novel drug candidates in hostdefense against all the infectious diseases and necrosis.

Examples of this invention have been shown in order to demonstrate thepotent anti-infectious roles of the plasminogen-activator system fromdifferent angles and in different models. Example 1 demonstrates thatplg−/− mice have much more severe tissue destruction and more severechronic inflammation as compared to plg+/+ mice after the induction ofbacterial arthritis. These plg−/− mice also have impairment in killingbacteria (Example 3) and lower levels of IL-10 expression (Example 9),but the infiltration of the infected joints by macrophages andneutrophils is not overfly impaired in plg mice (Example 4). Antibiotictreatment kills bacteria and reduces inflammation, but does not decreaseformation of necrotic tissue in plg^(−/−) mice (Example 2). However,systemic or local supplementation of plg^(−/−) mice with humanplasminogen (hPlg) restored the normal host defense against S.aureus-induced bacterial arthritis (Examples 5 & 6) and increased theIL-6 protein expression in the infected knee joints (Example 8), Localsupplementation of plg^(+/+) mice with human plasminogen enhances thehost defense against S. aureus infection (Example 7), which stronglyindicate that plasminogen is an excellent anti-infection agent superiorthan antibiotics and can be used effectively on wild-type normalanimals. The importance of the plasminogen-activation system in hostdefense and tissue remodeling against infection is further demonstratedby the use of uPA−/− mice (Example 10). Example 10 shows that factorsthat activate plasminogen can also be useful as a therapeutic sinceplasminogen appears to be less effective in the absence of an activator.Furthermore, the essential roles of plasminogen in the host defenseagainst infection were further confirmed by the use of plg−/− mice inanother bacterial arthritis model (i.v. injection of bacteria, Examples11 & 12) and another two open-wound infection models, incisional wound(Example 13) and scald bum wound (Example 14). Investigation of thespontaneous development of otitis media indicate that all of the plg−/−mice studied have ear infection whereas all of the plg+/+ mice remaineduninfected (Example 17). Bacterial recovery from the ear tissuesdemonstrated that only 1 type of bacteria identified in 1 out of 6plg+/+ mice, whereas 4 types of bacteria identified in 5 out of 6 plg−/−mice (Examples 15 & 16). Overall these examples have characterized thepluripotent roles of plasminogen from various anti-infectious aspectsand strongly support the conclusion that plasminogen and its derivativesare novel drug candidates in host defense against all the infectiousdiseases.

Since plasminogen compounds and methods of the present invention providean inflammatory response directed to infection or necrotic conditions,the compounds and methods of the present invention may provide a defenseagainst all infectious disease, especially bacterial infectious disease,and necrosis. Such infection conditions include infectious diseases andother diseases where tissue infection is commonly observed, for instanceduring infection defense, chronic ulcers and diabetic ulcers. Suchnecrosis exist not only in the disease model hereby studied, but alsoother types of diseases which can also induce tissue necrosis, such asavascular femoral head necrosis, papillary necrosis, hip osteonecrosis,renal cortical necrosis, acute tubular necrosis, acute retinal necrosis,acute tubular necrosis, myocardial infarction, pancreatic necrosis,ischemic colitis, necrotizing fascilitis. There are many causes ofnecrosis including injury, infection, cancer, infarction, invenomation,slow and non-healing wounds, diabetes and inflammation. In addition, itwas discovered that severe inflammation, tissue destruction, necrosisand bacterial growth all permanently persisted in plasminogen-deficientanimal, therefore offering a novel model for studying bacterialinfection, and tissue necrosis, and screening methods for identifyingand evaluating drugs and treatment methods for enhancing bacterialinfection and tissue necrosis.

Accordingly, in a first aspect the invention refers to the use of acompound that is a component of the plasminogen-activating pathway or acompound having the capacity to activate plasminogen either directly orindirectly via activating an upstream component of theplasminogen-activating pathway for the manufacture of a pharmaceuticalcomposition for the prophylaxis, prevention and/or treatment ofinfectious disease.

In a preferred embodiment the component of the plasminogen-activatingpathway is selected from plasminogen, human recombinant plasmin,Lys-plasminogen, Glu-plasminogen, plasmin, variants and analogues ofplasminogen and plasmin comprising one or more of the kringle andprotease domains of plasminogen and plasmin, mini-plasminogen,mini-plasmin, plasminogen activators, tPA and uPA.

In another preferred embodiment the compound with the capacity toactivate plasminogen is selected from streptokinase, saruplase,alteplase, reteplase, tenecteplase, anistreplase, monteplase,lanoteplase, pamiteplase, staphylokinase and recombinant forms andvariants of the components of the plasminogen-activating pathway.

In general, the component of the plasminogen-activating pathway or thecompound with the capacity to activate plasminogen can be administeredsystemically, locally, topically, intravenously, intramuscularly,subcutaneously, via inhalation, intrathecally, via local injection, viaintra-articular injection or per rectally. In a preferred embodiment,topical administration and/or local injection are used.

Also, the component of the plasminogen-activating pathway or thecompound with the capacity to activate plasminogen can be administeredin combination with a suitable polypeptide carrier such as albumin,gelatine, and the like and/or one or more stabilizing agent(s) such as adetergent, a cyclodextrin, a saccaride, dimethyl sulfoxide, glycerol,ethylene glycol, propylene glycol, an antioxidant, a metal chelator, anenzyme inhibitor and the like. Such additives can be used to improve thestability of the product in many ways, including by minimisingadsorption/absorption, by reducing aggregation, by improving solubility,by reducing oxidation and by reducing degradation. Methods for devisinga suitable carrier for a given protein are well known within the art.

Further, by way of example the component of the plasminogen-activatingpathway or the compound with the capacity to activate plasminogen may beadministered at a dose of 0.0001 to about 1 g, preferably 0.005 rag toabout 100 mg, more preferably from about 0.05 to about 50 mg. The dosein rag is with relation to square centimetre infected area (i.e.mg/square centimetre infected area)

Moreover, the administration of the component of theplasminogen-activating pathway or the compound with the capacity toactivate plasminogen may for example be repeated at least once,preferably at least every day.

In the context of the present invention and the claim scope the subjectcan be any mammal subject, especially a human subject.

Also, in another preferred embodiment the infectious disease is abacterial infectious disease.

Especially, the bacterial infectious disease is selected from otitismedia, bacterial arthritis. gingivitis, periodontitis, conjunctivitis,keratitis, wound infection, surgical wound infections, virginalinfections/injuries, necrosis, infections such as pneumonia, injuries inthe respiratory organs caused by burns and/or infections and infectedchronic leg ulcers in patients suffering from diabetes, systemicinfections, infections due to venous or combined venous/arterialinsufficiency or infectious arthritis or the like, infections secondaryto the injuries in the joint tissues caused by infections Especially, asillustrated by the appended examples, the invention is effective fortreating otitis media, bacterial arthritis, burn-related infection andincisional wound-related infection.

Staphylococcus aureus is the microorganism most frequently associatedwith bacterial arthritis, which results in synovial inflammation,cartilage and bone destruction, and eventually joint deformity.

Bacterial arthritis is a rapidly progressive and highly destructivejoint disease in humans. All destructive joint diseases, includinginflammatory disorders such as rheumatoid arthritis, are connected to anincreased incidence of bacterial arthritis. Certain forms of therapysuch as joint implants and immunosuppressive treatment display anincreased frequency of bacterial arthritis. S. aureus is the causativeagent in about 60% of nongonococcal bacterial arthritis cases. Inpatients with rheumatic diseases, this value is even higher, approaching75%. Laboratory models of bacterial arthritis have been used previously.In most instances bacteria have been injected intra-articularly.Morbidity and mortality due to S. aureus infections in the joints remainhigh despite the use of newer antibiotics. The increase in prevalence ofmultiantibiotic resistance in S. aureus is a major public healthconcern. Therefore, a novel, potent agent that can significantlyincrease the host defense is in great need.

Otitis media is the most common childhood disease except for commoncold. The most important etiological factor related to otitis media isbacterial or viral infections of the upper respiratory tract. Otitismedia is generally benign and a self-limiting disease, but despite this,the prescription rate of antibiotics is high. In fact, effects ofantibiotics in curing otitis media lack evidence and so far surgicalintervention is the therapy of choice for the treatment of recurrentacute otitis media (AOM) and otitis media with effusion (OME).

It is well known that the immediate colonization by the patient's normalskin flora (i.g. S. aureus and Streptococcus pyogenes) occurs followinginjury. Especially after the introduction of penicillin G in the early1950s, which resulted in the virtual elimination of Streptococcuspyogenes as a cause of infection in thermally injured patients, S.aureus became the principal etiological agent of wound infection.Therefore S. aureus is one of the most common bacterium species onopen-wound infection. Incisional wounds and burn wounds are the mostcommon wound types observed in clinical practice. Similar to thesituation in bacterial arthritis, due to the increasing problem of drugresistance of bacteria, a novel, potent agent that can significantlyincrease the host defense is in great need.

Basically, the invention is effective for treatment of all infectiousdiseases including infectious diseases induced by bacterial, viral andfungal infections.

Basically, since plasminogen compounds and methods of the presentinvention provide an inflammatory response directed to infection orinfectious disease or necrotic conditions, the compounds and methods ofthe present invention may provide an effective treatment of allinfectious diseases, particularly infectious diseases induced bybacterial, viral and fungal infections.

Bacterial and fungal agents that can cause infectious disease orsymptoms and that can be treated according the present inventioninclude, but are not limited to, the following Gram-Negative andGram-positive bacteria, bacterial families, and fungi: Actinornyees(e.g., Noreardia), Acinetobacter, Ciryvtococcus neoformans, Aspergillus,Bacillaceae (e.g., Bacillus anthrasis), Bacieroides (e.g., Bacteroidesfragilis), Blastomycosis, Bordetella, Borrelia (e.g., Borreliaburgdorferi), Brucella, Candidia., Campylobacter, Chlamydia, Clostridium(e.g., Clostridium botulinum, Clostridium dificile, Clostridiumperfringens, Clostridium tetani), Coccidioides, Corynebacterium (e.g.,Corynebacterium. diptheriae), Cryptococcus, Dermatocycoses, E. coli(e.g., Enterotoxigenic E. coli and Enterohemorrnagic E. coli),Enterobacter (e.g. Enterobacter aerogenes), Enterobacteriaceae(Klebsiella, Salmonella (e.g., Salmonella typhi, Salmonella enteritidis.Salmonella typhi), Serratia, Yersinia, Shigella), Erysipelothrix,Haemophilas (e.g., Haemophilus influenza type B), Helicobacter,Legionella (e.g., Legionella pneumophila), Leptospira, Listeria (e.g.,Listeria monocytogenes), Mycoplasma, Mycobacterium (e.g., Mycobacteriumleprae and Mycobacterium tuberculosis), Vibrio (e.g., Vibrio cholerae),Neisseriaceae (e.g., Neisseria gonorrhea, Neisseria meningitidis),Pasteurellacea, Proteus, Pseudomonas (e.g., Pseudomonas aeruginosa),Rickettsiaceae, Spirochetes (e.g., Treponema spp., Leptospira spp.,Borrelia spp.), Shigella spp., Staphylococcus (e.g., Staphylococcusaureus), Meningiococcus, Pneumococcus and Streptococcus (e.g.,Streptococcus pneumoniae and Groups A, B, and C Streptococci), andUreaplasmas.

These bacterial, parasitic, and fungal families can cause diseases orsymptoms, including, but not limited to: Sepsis such as bacteremia,hemorrhagic septicemia and fangemia; Central nervous system bacterialinfections such as Lyme neuroborreliosis, bacterial meningitis andencephalitis, cerebral toxoplasmosis and neurosyphilis; Bacterial eyeinfections, such as bacterial conjunctivitis, infectiouskeratoconjunctivitis, infectious keratitis, ocular tuberculosis, anduveitis; Bacterial ear infections such as otitis media, external otitis;Sexually transmitted diseases such as Chlamydia infections, gonorrhea,and syphilis; Infectious skin diseases such as cellulitis,dermatomycoses, and bacterial skin diseases such as actinomycosis,angiomatosis ecthyma, erysipelas, staphylococcal skin infections,cutaneous syphilis, and cutaneous tuberculosis; Bacterial vaginosis;Respiratory tract infections such as whooping cough and pneumonia suchas pneumococcal pneumonia, staphylococcal pneumonia and mycoplasmapneumonia; Urinary tract infections such as bacteriuria; Woundinfections such as surgical wound infections, chronic infected skinulcers, necrosis, open-wound infections; Bacterial arthritis; Infectiousbone diseases such as osteitis, osteomyelitis, periostitis, spondylitis,and osteartieular tuberculosis; Cardiovascular infections such asbacterial endocarditis, cardiovascular syphilis, and cardiovasculartuberculosis; Periodontal diseases such as gingivitis and periodontitis;AIDS-related opportunistic infections; Pelvic infections; Infectiouspregnancy complications; which accordingly can be treated according tothe present invention. For a more extensive listing of infectiousdiseases for which the invention would be effective, reference is madeto the webpagehttp://www.health.vic.gov.au/ideas/diseases/quicklinks.htm. or to anyrelevant review journal in the art disclosing infectious diseases (seereference list).

Since plasminogen-activating pathway and compounds of the presentinvention provide an inflammatory response to viral infections in asimilar manner as for bacterial infections and necrotic conditions, thecompounds and methods of the present invention provide a similarlyuseful defense against vital infections and conditions including thoselisted below.

Viral agents that can cause infectious disease or symptoms and that canbe treated according the present invention include, but are not limitedto, Arbovirus Infections, such as Bluetongue, Dengue, ArbovirusEncephalitis, Phlebotomus Fever, Rift Valley Fever, Tick-Borne Diseases,and Yellow Fever; Viral Bronchiolitis; Central Nervous System ViralDiseases, such as Encephalitis, Viral Meningitis, Myelitis,Poliomyelitis, and Pseudorabies; DNA Virus infection, such asAdenoviridae Infections, African Swine Fever, Circoviridae Infections,Hepadnaviridae Infections, Herpesviridae Infections including but notlimited to Herpes Simplex, Herpes Zoster, and CytomegalavirusInfections, Papillomavirus Infections, Parvoviridae Infections,Polyomavirus Infections, and Poxviridae Infections; Viral Encephalitis,such as Arbovirus Encephalitis, Herpes Simplex Encephalitis, andVaricella Zoster Encephalitis, Viral Eye Infections, such as viralConjunctivitis, Cytomegalovirus Retinitis, Herpes Zoster Ophthalmicusand Herpetic Keratitis; Viral Hepatitis, such as Hepatitis A. HepatitisB, Hepatitis C, Hepatitis D and Hepatitis E; Opportunistic Infections,such as AIDS-Related Opportunistic Infections; Viral Pneumonia; RNAVirus Infections, such as Arenaviridae Infections, AstroviridaeInfections, Birnaviridae Infections, Bunyaviridae Infections includingbut not limited to Hantavirus Infections, Caliciviridae Infections,Arbovirus Encephalitis, Flaviviridae Infections, Viral HemorrhagicFevers, Mononegavirales infections including; but not limited toRhabdoviridae Infections such as Rabies, Paramyxoviridae Infections suchas Morbillivirus infection including but not limited to measles,Pneumovirus Infections including but not limited to RespiratorySyncytial Virus Infection, and Rubulavirus Infections including but notlimited to mumps, Nidovirales Infections including but not limited toCoronavirus Infections such as Severe Acute Respiratory Syndrome,Orthomyxoviridae Infections, such as influenza, PicornaviridaeInfections, such as enterovirus infections, Reoviridae Infectionsincluding but not limited to Rotavirus Infections, RetroviridaeInfections including but not limited to Lentivurs Infections, andTogaviridae Infections; Viral Sexually Transmitted Diseases; Viral SkinDiseases, such as Erythema Infectiosum, Exanthema Subitum, HerpesSimplex, Molluscum Contagiosum, and Warts; Slow Virus Diseases, suchAIDS, Progressive Multifocal Leukoencephalopathy, and SubacuteSclerosing Panencephalitis; Tumor Virus Infections, such as Epstein-BarrVirus Infections, Marek Disease, and Papillomavirus Infections, andViremia.

In another embodiment, the composition comprises a combination of two ormore compounds which are components of the plasminogen-activatingpathway or compounds with the capacity to activate plasminogen.

In yet another embodiment, the composition further comprises at leastone antibiotic agent.

The antibiotic agent is e.g. selected from the group consisting oftetracyclines, amphenicols, beta-lactams, penicillins, sulphonamides,macrolides, lincosamides, streptogamins, streptomycins, quinolones andmetronidazoles, as well as any proper antibacterial agent, mycocide orfungicide.

Further, in yet another embodiment, the prophylaxis, prevention and/ortreatment of infectious disease comprises inducing an immune responseagainst an infectious pathogen.

In a second aspect, the invention relates to a method for theprophylaxis, prevention and/or treatment of infectious disease, whichcomprises administering a pharmaceutical composition comprising aneffective amount of a compound which is a component of theplasminogen-activating pathway or a compound with the capacity toactivate plasminogen to a subject in need of such treatment.

In still another aspect the invention relates to a pharmaceuticalcomposition for the prophylaxis, prevention and/or treatment ofinfectious disease comprising art effective amount of a compound whichis a component of the plasminogen-activating pathway or a compound withthe capacity to activate plasminogen, and a pharmaceutically acceptablecarrier.

In yet another aspect, the invention relates to a kit of parts for usein the prophylaxis, prevention and/or treatment of infectious diseasecomprising an effective amount of a compound which is a component of theplasminogen-activating pathway or a compound with the capacity toactivate plasminogen and at least one antibiotic, antiviral orantimycotic agent, in separate vials

In a further aspect, the invention relates to a method of identifying anagent that is useful in promoting host defense against infection,comprising the steps of: (a) administering a test agent to an animalhaving a bacterial arthritis; (b) evaluating at least one of thefollowing parameters: (i) the extent of bacteria, (ii) necrotic tissueformation, (iii) inflammatory cell activation, (iv) cytokine expressionof the infection e.g. bacterial arthritis by using fluorescent orradio-isotopic biomarker labels, microbiological plaque assay from bodyfluid or tissue homogenates, FACs analysis, ELISA, histologicalexaminations, and/or cytotoxicity assay (for killing bacteria, one cansimply recover bacteria from one tissue/organ, for necrotic tissueformation, one can quantify tissue autopsy, for inflammatory cellactivation, one can determine histochemically, ELISA, western blottingof different inflammatory cell markers and for cytokine expression,there are kits for the detection of the levels of cytokines. All ofthese methods are included in the Example section of this disclosurewhich can be used); (c) comparing the chosen parameter(s) of step (b)with a control value, wherein plasminogen can be used as a positivecontrol and a non-treated group as a negative control): and (d)selecting any test agent for which the chosen parameter(s) is(are) morebeneficial compared to the control value as an agent useful in promotinghost defense against infection.

In a preferred embodiment, the test of model animal is selected from amember of the group consisting of a wild-type animal and a transgenicanimal lacking endogenous expression of plasminogen.

In yet another aspect, the invention refers to a method for diagnosis ofan ongoing infection, comprising determining the diagnostic presence ofplasminogen.

In still another aspect, the invention relates to a kit for use in thedetermination of plasminogen in a sample from a patient, wherein thesample is from body fluids, serum, excreted waste products, such asurine or faeces, exhalation air, or the like, in order to determine anongoing infection and/or the effect of an ongoing treatment, comprisinga plasminogen determinant and means for collecting, storing and/orexamining the patient sample.

Accordingly, the present invention provides for a method of improvinghost defense against infection, comprising administering a compositioncomprising an active agent which is a component of theplasminogen-activation pathway or a compound with the capacity toactivate plasminogen. Preferably, the active agent is selected fromplasmin or plasminogen or an analogue of plasmin or plasminogen. Mostpreferably, the active ingredient is plasminogen. The active agent canbe administered by any route of administration known in the art.Preferred, non-limiting, routes of administration include topicalapplication and local injection. The agent may also be present in awound dressing applied onto the infected area, if possible, from whichit is transferred to the infected site. The agent may also be present ina rinsing solution, eye drops and gargling solution or the like, appliedto clean the infected area.

The invention also provides for a method of initiating the host defenseagainst infection in conditions where in host defense is retarded orimpaired, comprising administering an active ingredient which is plasminor plasminogen. In a particular embodiment, the method of the inventioncan be used for improving infection defense in conditions of local orsystemic low levels of plasmin or plasminogen. Such conditions may becongenital and/or acquired.

Examples of congenital conditions with systemic deficiency of plasmin orplasminogen include but are not limited to mutations in the plasminogen(PLG) gene (GenBank Reference Sequence accession No: NM_(—)000301,GeneID: 5340; the amino acid residue numbers herein refer to the maturehuman peptide as defined in GenBank accession No: NP_(—)000292)resulting in dysplasminogenias such as ALA601THR, VAL355PHE, SER572PROAND GLY732ARG, or in type I plasminogen deficiency such as ARG216HIS,TRP597TER, GLU460TER, LYS212DEL and LYS19GLU. In cases where congenitalplasminogen deficiency is present, administering a drug which isplasminogen is preferred.

Examples of acquired systemic and/or local defects of plasmin orplasminogen can be due to changes in physiologic states such aspregnancy, old age, stress, obesity, and temperature alterations.Various disease states, surgery, radiation, and diet can also triggermechanisms leading to impaired fibrinolytic, states. Several drugs,including anticancer agents, oral contraceptives, cytokines, and bloodcomponents can also produce transitory fibrinolytic deficit which canpredispose patients to thrombotic complications. The identification ofthe patient populations with an impaired fibrinolytic state is animportant step toward the prevention of thrombotic complications whichmay lead to such catastrophic events as myocardial infarction andthrombotic strokes. Both functional and immunologic methods havecurrently become available for the rapid diagnosis of fibrinolyticdeficit. Thus, it is important to evaluate patients who are at risk ofthrombotic complications due to fibrinolytic deficit.

In another embodiment, the invention provides a method for treatment ofinfection and enhancement of infection defense in human or non-humansubjects by administering a compound or drug which is plasminogen,plasmin, an activator of plasminogen, or a compound enhancing theproteolytic activity of plasmin.

In addition, the invention provides for a method of improving infectiondefense against bacterial arthritis and/or otitis media, comprisingadministering a composition comprising an active ingredient which is acomponent of the plasminogen activation pathway or a compound with thecapacity to activate plasminogen. In a preferred embodiment, the activeingredient is plasminogen, and the composition administered via localapplication.

Moreover, the invention provides for a method for reducing or preventingnecrosis formation by administering a composition comprising local orsystemic administration of a composition comprising a compound which isa component of the plasminogen activation pathway or a compound with thecapacity to activate plasminogen. The composition may be part of a gel,lotion, balm, paste, wound bandage, or wound dressing. Alternatively,the composition may be administered systemically. In one embodiment, themethod of the invention is applied in conjunction with plastic surgeryto reduce the occurrence and the formation of infection, ulcer andnecrosis.

In yet another embodiment, the invention provides for a method fortreatment of infection and enhancement of infection defense in subjectswith defects in plasmin-plasminogen system activity by administering acompound or drug which is plasminogen, plasmin, an activator ofplasminogen, or a compound enhancing the proteolytic activity ofplasmin.

The invention further provides for a method of identifying a compounduseful for improving infection defense in an animal model. According tothe method of the invention, an infection is inflicted upon aplasminogen-deficient animal, and the test compound administered to theanimal via a predetermined route. Infection defense in theplasminogen-deficient animal is then compared to a control value suchas, e.g., infection defense in a wild-type animal, to evaluate whetherthe test compound improved the rate of infection, defense or reducednecrosis formation. One animal model preferred for this method isknock-out mice lacking one or both alleles of plasminogen, or transgenicmice. In one embodiment, the knee joints are infected, and the hostdefense of the joints is studied in the presence and absence of addedplasminogen. In another embodiment, the spontaneous development ofotitis media is followed. The gross appearance of the TM was carefullyexamined and documented under an otomicroscope, and the host defense ofthe TMs is studied in the presence and absence of added plasminogen. Inyet another embodiment, an open wound, for instance burn and incisionalwounds, is infected and the host defense against infection at thewounded site is studied in the presence and absence of addedplasminogen.

Furthermore, the invention provides for a method for in vivo screeningfor drugs that can be used for improving host defense against infectioncomprising an in vitro or in vivo model in which plasminogen isexpressed. The in vivo model comprises an animal that is wild-type orplasminogen-deficient. After administration of one or more drugs to bescreened, the activity or levels of plasminogen and/or plasmin will bemeasured. In a preferred embodiment, the animal model is a bacterialarthritis model in the knee joints, and bacterial arthritis is inducedbefore, in conjunction with, or after the administration of the drug. Inanother embodiment, the animal model is open-wound infection model andthe open-wound infection is induced before, in conjunction with, orafter the administration of the drug.

The invention also provides for a method for improving host defenseagainst infectious diseases, by administering a composition comprisingan activator of plasminogen activity, or a compound mimic of plasminogenexpression. Preferably, plasminogen is administered locally to attain ahigh concentration in the infected area. In another embodiment, thecomposition comprises a compound that mimics plasminogen/plasminactivity and molecules with similar activity. In still anotherembodiment, the composition comprises a drug which up-regulates theexpression of plasminogen.

Additionally, the invention provides for a method of treating a chronicinfection and necrosis by administering a drug that up-regulates theexpression of plasminogen or plasminogen-activators.

Still further, the invention refers to the use f a compound of the groupcomprising: plasminogen, plasmin, a fragment of plasminogen or plasmin,a component of the plasminogen activation pathway, a plasminogenanalogue, a plasmin analogue, or an analogue of a component of theplasminogen activation pathway, or a compound with the capacity toactivate plasminogen for the manufacture of a medicament for promotinghost defense of bacterial arthritis and otitis media and open woundinfection, and/or for removing necrotic tissue and/or for improving hostdefense against infection and/or for reducing necrotic tissue formationin a healing wound.

BRIEF DESCRIPTION OF THE DRAWINGS

The above features and many other advantages of the invention willbecome better understood by reference to the following detaileddescription when taken in conjunction with the accompanying drawings.

FIG. 1A-H. Histologies of representative sections of arthritic kneejoints from plg^(+/+) (left) and plg^(−/−) (right) mice. Plg^(+/+) andplg^(−/−) mice were given an intraarticular injection of 1×10⁶ CFU S.aureus. (A, C, E): Arthritic knee joints front plg^(+/+) mice at days 7,14, and 28, respectively, after arthritis induction. (B, D, F):Arthritic knee joints from plg^(−/−) mice at days 7, 14, and 28,respectively, after arthritis induction. (G): Arthritic knee joints fromplg^(+/+) mice 7 days after antibiotic treatment. (H): Arthritic kneejoints from plug mice 7 days after antibiotic treatment. Necrotic tissueis observed in the joint cavity (arrow). Synovial membrane (Sm).

FIG. 2. Quantification of necrotic tissue in the infected joints at eachtime point. The amount of necrotic tissue in the infected joints wasscored histologically, as described in Materials and Methods, Plg^(+/+)and plg^(−/−) mice were compared on day 7, day 14 and day 28 after 1×10⁶CFU of S. aureus injection. Results are expressed as mean±SD. **P<0.01,by student t test.

FIG. 3. Neutrophil and macrophage numbers in the infected knee joints ofplg^(+/+) and plg^(−/−) mice that were given an intraarticular injectionof 1×10⁶ CFU of S. aureus. A. Infiltrated neutrophil numbers werecompared on day 1, day 7 and day 14 ‘idler bacterial injection. Barsrepresent mean value of 5 mice. B. Infiltrated macrophage numbers werecompared on day 1, day 7 and day 14 after bacterial injection. Barsrepresent mean value of 5 mice. Error bars show SDs.

FIG. 4A-F. Histological analysis of whole knee joint from plg^(+/+) andplg^(−/−) mice after plasminogen supplementation. A, B: Controlarthritic knee joints from plg mice on days 7 and 14. respectively,after bacterial injection. C, D: Control arthritic knee joints (injectedwith PBS) from plg^(−/−) mice on days 7 and 14, respectively, afterbacterial injection. E: Plg^(−/−) arthritic knee joints supplementedwith human plasminogen (hPlg) from day 0 to day 7 after bacterialinjection. F: Plg^(−/−) arthritic knee joints supplemented with humanplasminogen from day 7 to day 14 after bacterial injection.

FIG. 5A-C. IL-6 protein expression levels in the synovium.Immunostaining of representative sections of knee joints from plg^(+/+)and plg^(−/−) mice. A: Control arthritic knee joints treated with PBSfrom plg^(+/+) mice on day 7 after bacterial injection. B: Controlarthritic knee joints treated with PBS from plg^(−/−) mice on day 7after bacterial injection. C: Plg^(−/−) arthritic knee jointssupplemented with human plasminogen (hPlg) from day 0 to day 7 afterbacterial injection. Pink color shows the IL-6 in the synovium (arrow).

FIG. 6A-B. Western blot analysis of the expression level of IL-10protein in uninfected and infected knee joints. A, levels of IL-10 inthe lysates. Lane 1: uninfected knee joint lysates of plg^(−/−) mice;Lane 2: uninfected knee joint lysates of plg^(+/+) mice; Lane 3:plg^(−/−) arthritic knee joints lysates at day 3 after bacterialinjection; Lane 4: plg^(+/+) arthritic knee joints lysates at day 3after bacterial injection; Lane 5: plg^(−/−) arthritic knee jointslysates at day 7 after bacterial injection: Lane 6: plg^(+/+) arthriticknee joints lysates at day 7 after bacterial injection. B, Levels ofβ-actin in the lysates correspondent to each lane in A as control.Experiments were repeated at least 3 times and representative resultswere shown.

FIG. 7A-F. Morphology of representative middle ear sections fromwild-type mice and plg-deficient mice, stained with toluidine blue (Aand B) and immunohistochemical stainings for fibrin (C and D and keratin(E and F). A, The middle ear from a wild-type mouse. No effusionmaterial is detected in the middle ear cavity (MEC). B. The middle earfrom a plg-deficient mouse. Otitis media is present in the MEC. C and D.The middle ears from a wild-type mouse and plg-deficient muserespectively, analyzed by immunohistochemical staining for fibrin(ogen).E and F. The middle ears from a wild-type mouse and plg-deficient mouserespectively, analyzed with immunohistochemical staining for keratin. O,ossicle. Bar 50 μm.

FIG. 8A-H. Immunohistochemical stainings for T cells, B cells,macrophages and neutrophils in middle ears of wild-type andplg-deficient mice. Middle ears from a wild-type control (A, C, E, G)and a representative plg-deficient mouse (B, D, F, H) were analyzed byimmunohistochemical stainings of T cells (A and B), B cells (C and D),macrophages (E and F) and neutrophils (G and H). O, ossicle. Bar 50 μm.

FIG. 9. Bacterial numbers in knee joints of and plg−/− and plg+/+ micewith different local and systemic treatments after inoculation of 1×10⁶CFU of S. aureus Phillips at the knee joints.

FIG. 10. Bacterial numbers in knee joints of plg+/+ mice after localinjection with Plg (closed box) or PBS (open box) 3 days afterinoculation of S. aureus at the knee joints. Note in wild-type micelocally injected with Plg the bacterial number is significantly loweredfor 5 folds than that of wild-type locally injected with PBS.

FIG. 11. Bacterial numbers in knee joints of uPA-deficient and wild-typemice. Note in wild-type mice the bacterial number quickly subsided afterthe inoculation of S. aureus at day 0, whereas in uPA-deficient mice thenumber of bacteria were constantly over 2.0×10⁵ CFU throughout theexperimental period.

FIG. 12A-F. Histologies of representative sections of arthritic kneejoints from uPA-deficient (uPA^(−/−), left) and wild-type (uPA^(+/+),right) mice at days 7 (A, D), 14 (B, E) and 28 (C, F) after anintraarticular injection of 1×10⁶ CFU S. aureus. Note in uPA−/− micethere are much more edema, tissue destruction and necrotic tissueformation throughout the experiment, whereas in uPA+/+ mice theinflammation was just transiently present at day 7 after arthritisinduction and subsided thereafter.

FIG. 13: Comparison of body weight changes between plg^(+/+) andplg^(−/−) mice. Time course of body weight changes after 1×10⁶ CFU ofbacterial injection. Mice body weight was checked every 24 hr, from day1 to day 21.

FIG. 14. Severity of septic arthritis. Results from the evaluation ofseverity in septic arthritis using arthritic index as described inmaterials and methods. Arthritis was induced, by 1×10⁶ CFU S. aureusPhillips injected intravenously. Plg^(+/+) mice (n=15) and plg^(−/−)mice (n=16). For each time point, mean±SEM are shown. Statisticalsignificance test was performed by using the Mann-whitney u-test(deficient mice versus control mice). *P<0.05 was consideredsignificant.

FIG. 15A-H: Plasminogen deficiency exacerbates histological features ofseptic arthritis. Histologies of paw joints sections stained withSafranin-O. Morphology of the representative sections of arthritic pawjoints after intravenous injection of 1×10⁶ CFU S. aureus in plg^(+/+)(left) and plg^(−/−) (right) mice Arthritic paw joints from wild-typemice on days 1 (A), 3 (B), 7 (C) and 14 (D) after arthritis onset.Arthritic paw joints from plg^(−/−) mice on days 1 (E), 3 (r), 7 (G) and14 (H) after arthritis onset.

FIG. 16A-B: Necrotic tissue in infected ankle joints. Necrosis was baseon histological observation. Some samples that were identified asnecrosis were further confirmed by TUNEL staining.

FIG. 17A-B: Fibrin deposition in the infected ankle joints.Immunohistochemical detection of fibrin in arthritic knee joints.Paraffin-embedded tissue sections were stained with a rabbit anti-murinefibrin(ogen) antibody. Brown color indicates positivity. Arthritic kneejoint on day 14 in plg^(+/+) (left) and plg^(−/−) (right) mice afterarthritis onset. Similar fibrin deposition in the plg^(−/−) andplg^(+/+) infected joints.

FIG. 18. Bacterial numbers in wound tissue of plg−/− mice with localtreatments of plg or PBS after induction of incisional wound inoculatedlocally with 1×10⁷ CPU of S. aureus Phillips in the dorsal skin.

FIG. 19A-B. Representative appearance of plg−/− mice with localtreatments of plg or PBS after induction of incisional wound inoculatedlocally with 1×10⁷ CFU of S. aureus Phillips in the dorsal skin.

FIG. 20 Bacterial numbers in wound tissue of plg−/− mice with localtreatments of plg or PBS after induction of burn wound inoculatedlocally with 1×10⁶ CFU of S. aureus Phillips at the scald skin.

DEFINITIONS

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow, or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them.

“A compound of the group comprising: plasminogen, plasmin, a componentof the plasminogen activation pathway, a plasminogen analogue, such asmini-plasmin, a plasmin analogue, an analogue of a component of theplasminogen activation pathway, a plasminogen activator” refers to acompound that directly or indirectly provides the effect of plasminogenor plasmin, respectively.

“A component of the plasminogen activation pathway” refers toplasminogen. Lys-plasminogen, Glu-plasminogen, variants and analogues ofplasminogen comprising one ore more domains of plasminogen such as oneore more of the kringle domains and the proteolytic domain exemplifiedby mini-plasminogen; plasmin and variants and analogues of plasmincomprising at least one ore more domains of plasmin such as one or moreof the kringle domains and the proteolytic domain, exemplified bymini-plasmin and delta-plasmin; a plasminogen activator having the finaleffect of activating plasminogen, e.g. by a cascade of events resultingin the formation or activation of plasminogen exemplified by uPA and tPAand variants and analogues of tPA and uPA comprising one ore moredomains of tPA or uPA such as one ore more of the kringle domains andthe proteolytic domain. Variants of plasminogen, plasmin, tPA and uPAinclude all naturally occurring genetic variants of human as well asother mammalian forms of these proteins, as wells as mutant variants ofthese proteins obtained by conservative amino acid replacements. An“analogue” of plasminogen or plasmin is a compound providing essentiallyan analogous effect as plasminogen or plasmin, respectively, as measuredby enzymography, ELISA (enzyme-linked immunosorbent assay) and FACS(fluorescence activated cell sorter), There is also an assay formeasuring levels of converted plasmin activity as described previously:Ny, A., Leonardsson, G., Hagglund, A. C., Hagglof, P., Ploplis, V. A.,Carmeliet, P., and Ny, T. (1999). Ovulation in plasminogen-deficientmice. Endocrinology 140, 5030-5035.). An “analogue” of a component ofthe plasminogen activation pathway is a compound providing essentiallyan analogous effect as a component of the plasminogen activation pathwayas measured by the levels or plasmin activity that this analogueactivates.

“Necrosis” refers to death of tissue in the body. This happens when notenough blood is supplied to the tissue, whether from injury, radiation,or chemicals. Necrosis is not reversible. There are many causes ofnecrosis including injury, infection, cancer, infarction, invenomation,chronic wounds, ulcers and inflammation.

“Topical” and “topical application” refer to non-systemic, local,administration of an active ingredient. Thus, topical application canrefer to application of an active ingredient to the external surface ofthe interesting area.

“Local injection” refers to non-systemic, local administration of anactive ingredient into tissue of/nearby the interested area.

“Intra-articular injection” refers to local administration of an activeingredient into the joint space between two connecting bones.

“Infectious diseases” and “infection” refers to the detrimentalcolorization of a host organism by a foreign species. In an infection,the infecting organism seeks to utilize the host's resources to multiply(usually at the expense of the host). The infecting organism, orpathogen, interferes with the normal functioning of the host and canlead to chronic wounds, gangrene, loss or an infected limb, and evendeath. The host's response to infection is inflammation. Colloquially, apathogen is usually considered a microscopic organism, the most familiarare viruses and bacteria. Other infectious pathogens are viroids andeukaryotic organisms, ranging from single-celled fungi and protozoa,through large complex metazoan such as parasitic worms.

The “activity” of a protein or compound refers to the effect that theprotein or compound has on a specific reaction, and is a measure of itsability to affect, modulate, participate in, or promote the reaction.Generally, the activity of a protein or other compound can be measured.For example, in the case of enzymes such as plasmin, PA, and MMPs, andmodulators enzyme activity can he expressed as the rate at which theproduct of the reaction is produced, represented, e.g., as the tanamount of product produced per unit of time and of enzyme (e.g.,concentration or weight). In the case of modulators such as PAI-1 oruPA, activity cart refer to the ability of the modulator to inhibit orpromote, increase or decrease, up- or down-regulate, the rate of areaction or the amount of product farmed from the reaction.

A “wound” is a break in the structure of an organ or tissue, includingepithelium, connective tissue, and muscle tissue, caused by an externalagent. Examples of wounds include, but are not limited to, bruises,grazes, tears, cuts, punctures, and burns. A particular type of woundsare those that are a consequence of plastic surgery procedures.

“Otitis media” is defined as inflammatory conditions of the ear. Otitismedia,including acute otitis media (AOM) and otitis media with effusion(OME), is the most common childhood disease except for common cold (5).The most important etiological factor related to otitis media isbacterial or viral infections of the upper respiratory tract. Thebiochemical composition of the middle ear effusions in otitis mediareflects inflammatory changes in the middle ear mucosa. The fluid is amixture of transudates and secretory products from glands as well asproducts from inflammatory cells and bacteria.

“Treatment” of a subject, or “treating” a subject for a disease orcondition herein means reducing or alleviating clinical symptoms of thedisease or condition such as impaired or slow wound-healing.

“Enhancing” wound healing means increasing the speed by the which thewound heals. Alternatively, “enhancing” wound healing means reducing theformations of scar tissue during or after healing.

A “subject” herein includes both human and non-human animals. Non-humananimals include, without limitation, laboratory animals such as mice,rats, rabbits, hamsters, guinea pigs, etc.; domestic animals such asdogs and cats; and farm animals such as sheep, goats, pigs, horses, andcows. A non-human animal of the present invention may be a mammalian ornon-mammalian animal; a vertebrate or an invertebrate.

A “control”, “control value” or “reference value” in an assay is a valueused to detect an alteration in, e.g., the healing of a skin wound, orhealing of a perforated tympanic membrane, or any other assays describedherein. For instance, when studying healing of a tympanic membraneperforation, the inhibitory/stimulatory effect of an agent can beevaluated by comparing the healing of a wound or perforation to that ofa control. The control or reference may be, e.g., a predeterminedreference value, or may be determined experimentally. In such an assay,for example, a control or reference may be the healing of a similarwound or perforation in an animal not exposed to the drug or activeagent.

A subject “at risk for”, “predisposed to”, or “susceptible to” a diseaseor condition means that the risk fir the individual to contract ordevelop the disease or condition is higher than in the averagepopulation.

A “deficiency” of a compound means that the amount, level, orconcentration of the compound is significantly lower than a controlvalue. For example, in a plasminogen-deficient animal, the body fluidand tissue levels of plasminogen is significantly lower than in awild-type animal.

As used herein, “about” or “approximately” shall mean within 50 percent,preferably within 20 percent, more preferably within 5 percent, of agiven value or range.

A value which is “substantially different” from another value can meanthat there is a statistically significant difference between the twovalues. Any suitable statistical method known in the art can be used toevaluate whether differences are significant or not. A “statisticallysignificant” difference means a significance is determined at aconfidence interval of at least 90%, more preferably at a 95% confidenceinterval.

Molecular Biology Definitions

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al. (Molecular Cloning—ALaboratory Manual, Cold Spring Harbor Laboratory Press, 1989): Glover(DNA Cloning: A Practical Approach, Volumes I and II, 1985); :Flames andHiggins (Nucleic Acid Hybridization, 1985); Hames and Higgins(Transcription And Translation, 1984); Freshney (Animal Cell Culture,1986); Perbal (A Practical Guide To Molecular Cloning, 1984); andAusubel et al. (Current Protocols in Molecular Biology, John Wiley &Sons, Inc., 1994).

If appearing herein, the following terms shall have the definitions setout below.

A “protein” or “polypeptide”, which terms are used interchangeablyherein, comprises one or more chains of chemical building blocks calledamino acids that are linked together by chemical bonds called peptidebonds.

An “enzyme” means arty substance, preferably composed wholly or largelyof protein, that catalyzes or promotes, more or less specifically, oneor more chemical or biochemical reactions. The term “enzyme” can alsorefer to a catalytic polynucleotide (e.g. RNA or DNA). A “test” enzymeis a substance that is tested to determine whether it has properties ofan enzyme.

A “native” or “wild-type” protein, enzyme, polynucleotide, gene, orcell, means a protein, enzyme, polynucleotide, gene, or cell that occursin nature.

A “mutant”, “altered”, “variant” or “modified” protein, enzyme,polynucleotide, gene, or cell, means a protein, enzyme, polynucleotide,gene, or cell, that has been altered or derived, or is in some waydifferent or changed, from a parent protein, enzyme, polynucleotide,gene, or cell. An alteration in a gene includes, but is not limited to,alteration the promoter region, or other regions which affecttranscription, which can result in altered expression levels of aprotein. A mutant or modified protein or enzyme is usually, although notnecessarily, expressed from a mutant polynucleotide or gene.

A “mutation” or “alteration” means any process or mechanism resulting ina mutant protein, polynucleotide, gene, or cell. This includes anymutation in which a protein, polynucleotide, or gene sequence isaltered, any protein, polynucleotide, or gene sequence arising from amutation, any expression product (e.g. protein) expressed from a mutatedpolynucleotide or gene sequence, and any detectable change in a cellarising from such a mutation.

“Function-conservative variants” are proteins or enzymes in which agiven amino acid residue has been changed without altering overallconformation and function of the protein or enzyme, including, but notlimited to, replacement of an amino acid with one having similarproperties (such as for example, acidic, basic, hydrophobic, and thelike) “conservative amino acid replacements”. Amino acids with similarproperties are well known in the art. For example, arginine, histidineand lysine are hydrophilic-basic amino acids and may be interchangeable.Similarly, isoleucine, a hydrophobic amino acid, may be replaced withleucine, methionine or valine. Amino acids other than those indicated asconserved may differ in a protein or enzyme so that the percent proteinor amino acid sequence similarity between any two proteins of similarfunction may vary and may be, for example, from 70% to 99% as determinedaccording to an alignment scheme such as by the Cluster Method, whereinsimilarity is based on the MEGALIGN algorithm. A “function-conservativevariant” also includes a polypeptide or enzyme which has at least 60%amino acid identity as determined by BLAST or FASTA algorithms,preferably at least 75%, most preferably at least 85%, and even. morepreferably at least 90%, and which has the same or substantially similarproperties or functions as the native or parent protein or enzyme towhich it is compared.

The Plasminogen-Activation System

Plasmin is the key component of the PA system. It is a broad-spectrumprotease which has the ability to degrade several components of the ECMincluding fibrin, gelatin, fibronectin, laminin and proteoglycans (6).In addition, plasmin can convert some pro-matrix metalloproteinases(pro-MMPs) to active MMPs. It has therefore been suggested that plasminmay be an important upstream regulator of extracellular proteolysis.Plasmin is formed horn the zymogen plasminogen through proteolyticcleavage by either of two physiological PAs, tPA or uPA. As plasminogenis present in plasma and other body fluids at relatively high levels,the regulation of the PA system occurs mainly at the level of synthesisand activity orate PAs. Synthesis of the components of the PA system ishighly regulated by different factors such as hormones, growth factorsand cytokines. In addition, there exist specific physiologicalinhibitors of plasmin and PAs. The main inhibitor of plasmin isα₂-antiplasmin (7). The activity of PAs is regulated by PAI-1, whichinhibits both uPA and tPA, and PAI-2, which inhibits mainly uPA. Certaincells also have a specific cell-surface receptor for uPA (uPAR) that candirect proteolytic activity to the cell surface.

Plasminogen is a single-chain glycoprotein consisting of 791 amino acids(mature human peptide, GenBank. Accession No: NP_(—)000292) with amolecular mass of approximately 92 kDa (8; 9). Plasminogen is mainlysynthesized in the liver and is abundant in most extracellular fluids.In plasma the concentration of plasminogen is approximately 2 μM.Plasminogen therefore constitutes a large potential source ofproteolytic activity in tissues and body fluids.

Plasminogen exists in two molecular forms: Glu-plasminogen andLys-plasminogen. The native secreted and uncleaved form has anamino-terminal (N-terminal) glutamic acid and is therefore designatedGlu-plasminogen. However, in the presence of plasmin. Glu-plasminogen iscleaved at Lys⁷⁶-Lys⁷⁷ to become Lys-plasminogen. Compared toGlu-plasminogen, Lys-plasminogen has a higher affinity for fibrin and isactivated by PAs at a higher rate. These two forms of plasminogen can becleaved at the Arg⁵⁶⁰-Val⁵⁶¹ peptide bond by uPA or tPA, resulting inthe formation of the disulphide-linked two-chain protease plasmin. Theamino-terminal part of plasminogen contains five homologoustriple-loops, so-called kringles, and the carboxyl-terminal partcontains the protease domain. Some of the kringles containlysine-binding sites which mediate the specific interaction ofplasminogen with fibrin and its inhibitor α₂-AP. A novel and interestingfinding is that a 38-kDa fragment of plasminogen, consisting of kringles1-4, is a potent inhibitor of angiogenesis. This fragment is termedangiostatin and can be generated from plasminogen through proteolyticcleavage by several MMPs.

The main substrate for plasmin is fibrin, and dissolution of fibrin ispivotal for prevention of pathological blood clot formation (10).Plasmin also has substrate specificities for several other components ofthe ECM, including laminin, fibronectin, proteoglycans and gelatin,indicating that plasmin also plays an important role in ECM remodeling.Indirectly, plasmin can also degrade additional components of the ECMthrough its ability to convert some pro-MMPs to active MMPs, includingMMP-1, MMP-2, MMP-3 and MMP-9. It has therefore been suggested thatplasmin may be an important upstream regulator of extracellularproteolysis.

Models of Bacterial Arthritis to Study Infection

Staphylococcus aureus is the micro-organism that is most frequentlyassociated with bacterial arthritis, which results in synovialinflammation, cartilage and bone destruction, and eventually jointdeformity (11). Various animal species including mammals, birds andreptiles have been observed to develop spontaneous S. aureus arthritisand are therefore potential models for the induction of the disease.Considering the route of how the staphylococci spread through the bodyto reach the joints, which is an important trait, it has been clearlyshown that the great majority of bacterial joint infections in humansare spread hematogenously. Thus, the optimal way to deliver livebacteria to provide a model of infection is via intravenous (i.v.)injection. On the other hand, the intra-articular route of bacterialinoculation bypasses the early stages of pathogenesis, and thereforeprovides a more defined model of bacterial arthritis. Thus in thepresent studies, the intra-articular way to deliver bacteria in order tobetter study the local bacterial growth, tissue destruction, necrotictissue formation and inflammation has mainly been used as the model.However, we also performed a series of bacterial arthritis study usingS. aureus i.v. injection. Data obtained from both models have showncomparable results and both supported the conclusion thatplasminogen/plasmin is essential in the host defense against S.aureus-induced bacterial arthritis.

It is well known that the immediate colonization by the patient's normalskin flora (i.g. S. aureus and Streptococcus pyogenes) occurs followinginjury. Especially after the introduction of penicillin G in the early1950s, which resulted in the virtual elimination of Streptococcuspyogenes as a cause of infection in thermally injured patients, S.aureus became the principal etiological agent of wound infection.Therefore S. aureus is one of the most common bacterium species onopen-wound infection. Incisional wounds and burn wounds are the mostcommon wound types observed in clinical practice. Therefore, in thecurrent patent application, the open-wound infection models we have usedare infections by S. aureus, the principal etiological agent of woundinfection, on burn and incisional wounds, the most common wound types inpractice. Therefore, we consider the data obtained from these two openwound infection models give very important indication to the feasibilityof applying the knowledge to the clinical situation.

EXAMPLES

The invention is further described by means of the following examples.However, these examples are only illustrative of the invention, and inno way limits the scope and meaning of the invention. Indeed, manymodifications and variations of the invention will be apparent to thoseskilled in the art upon reading this specification, and can be madewithout departing from its sprit and scope.

Example 1

Persistent Inflammation and Tissue Destruction in plg^(−/−) Mice DuringS. aureus-Induced Bacterial Arthritis

This Example shows that plasminogen-deficient mice had persistentinflammation and tissue destruction compared to wild type controlsiblings. There are significantly more severe histopathological changesin plg^(−/−) mice than in plg^(+/+) mice during S. aureus-inducedbacterial arthritis.

Methods

Mice. Plasminogen-heterozygous (plg^(+/+)) mice of a mixed geneticbackground (129×C57BL/6) were intercrossed to generate plg^(+/+),plg^(+/+) and plg^(−/−) mice. Male plg^(+/+) and plg^(−/−) mice at 8-12weeks of age were used for the experiments (Ploplis V A, Carmeliet P,Vazirzadeh S, Van Vlaenderen I, Moons L, Plow E F, Collen D: Effects ofdisruption of the plasminogen gene on thrombosis, growth, and health inmice. Circulation 1995, 92:2585-7593).

Induction of bacterial arthritis. Bacterial strain used in the study wasS. aureus Phillips (Courtesy from. Dr. Höök, Department of rheumatologyand Clinical immunology, Gothenburg University, Sweden). Arthritis wasinduced by local injection of 1×10⁶ colony-forming units (CFU) of S.aureus Phillips in 10 μl sterile PBS into the right knee joints of mice.As controls, the left knee joints were injected with 10 μl sterile PBSalone. Mice were sacrificed at different time points after inoculationand samples were taken to evaluate the severity of the disease. TheRegional Ethical Committee of timed University approved all experimentalprotocols.

Histological analysis. At days 7, 14, and 28 after bacterial injection,mice were sacrificed and samples of whole knee joints were collected forhistological analysis. In brief, knee joints were first fixed in 4%paraformaldehyde, embedded in paraffin, and thereafter 8-μm sectionswere prepared. Slides containing tissue sections were stained withSafranin-O for histological analysis. At least 10 knee joints wereincluded in each experimental group.

Quantification of necrotic tissue in the infected joints. The images ofthe knee joints histological sections were taken with a Leica DC300Fdigital camera attached to a Leica DM LB microscope (Leica, Wetzlar,Germany). For determination of the amount of necrotic tissue in theinfected joints, the images of entire knee joints histological sectionswere divided into 50×40 grids at ×50 magnification. Each square withinthe grid that contained the necrotic tissue was counted as a ‘hit’.Three independent, randomly selected sections were counted from eachjoint and 5 joints from separate mice of the same genotype were used ateach time point. Mean values of the ‘hit’ were calculated and shown.

Results

To study the effects of plasmin on the clinical outcome of bacterialarthritis, mice were injected with 1×10⁶ CFU of S. aureus Philips in theknee joints. Thereafter, mice were sacrificed at different time pointsand the knee joints were dissected. Macroscopic examinations of kneejoint samples from days 7, 14, and 28 after intra-articular injectionrevealed that plg^(+/+) mice had similar levels of slight swelling inthe knee joints. However, in plg^(−/−) mice, the sizes of the bacterialinjected knee joints increased during the whole experimental period. Atday 28, the knee joints with bacterial injected were significantlyenlarged, the joint cavities were filled with purulent material, and thesynovial surfaces strongly bulged out.

The bacterial injected knee joints of plg^(+/+) and plg^(−/−) mice atdays 7, 14 and 28 were prepared for histological analysis. As shown inFIG. 1, considerably more severe arthritis was observed in plg^(−/−)mice than in plg^(+/+) mice at all observation time points. At day 7 inplg^(+/+) mice, inflammatory cells had infiltrated the joint cavity, thesynovial membrane was thicker than normal, and the cartilage surface hadbeen degraded (FIG. 1A, Sm). At day 14, inflammatory cells were observedadjacent to the eroded bone (FIG. 1C, arrow). However, at day 28, thenumber of inflammatory cells had declined and tissue repair of thedamaged cartilage and bone had started (FIG. 1E). The diseasedevelopment pattern was completely different in plg^(−/−) mice. At day 7in plg^(−/−) mice, large amount of inflammatory cells had infiltratedthe joint cavity, several parts of the bone were eroded, and necrotictissue was observed (FIG. 1B). At day 14, the synovial membrane hadbecome thicker than that at day 7, and the necrotic area had increased.Cartilage degradation and bone erosion were much more severe than at day7 (FIG. 1D). At day 28, the whole knee joint was almost completelydegraded, with only necrotic tissue and small parts of the cartilageremained (FIG. 1F). Semi-quantitive studies of the samples sectionsindicated thin plg^(−/−) mice had significantly higher levels of thetissue necrosis than that in plg^(−/−) mice during whole diseasedevelopment (FIG. 2). These data indicate that there are significantlymore severe histopathological changes in plg^(−/−) mice than inplg^(+/+) mice during S. aureus induced bacterial arthritis.

Example 2

Antibiotic Treatment Kills Bacteria and Reduces Inflammation, But DoesNot Decrease Formation of Necrotic Tissue in plg^(−/−) Mice

Methods

This experiment with performed in a similar manner as Example 1, exceptfor administration of antibiotics to some of the animals.

Antibiotic treatment. The antibiotic cloxacillin (AstraZeneca,Södertälje, Sweden) was dissolved in sterile PBS and injectedintraperitoneally into mice at a dosage of 0.5 mg/g body weight every12-hour, starting at day 7 after bacterial injection. Mice were killedat day 14 after bacterial injection.

Results

The effects of antibiotic treatment on disease development in plg^(+/+)and plg^(−/−) mice were also investigated. Mice were injected withbacteria at day 0 and treated with cloxacillin twice per day from day 7to day 14 after bacterial injection. Recovery of bacteria from theinfected joints at day 14 indicated that the bacteria were completelykilled in plg^(−/−) mice alter cloxacillin treatment (data not shown).Histopathological analysis revealed that the inflammation had subsidedin plg^(+/+) mice antibiotic treatment (FIG. 1G) compared to mice thathad not been treated with cloxacillin (FIG. 1C). In plg^(−/−) micetreated with cloxacillin, the levels of inflammation were still higherthan that in plg^(+/+) mice with the same treatment. The cartilage andhone had largely been repaired (FIG. 1H). Noticeably, small areas ofnecrotic tissue remained in the synovium (arrow, FIG. 1H). These datareveal that intraperitoneal administration of cloxacillin successfullyeliminated bacteria from knee joints, reduced tissue destruction andrestored tissue repair processes in plg^(−/−) mice. However, large areaof necrotic tissue remained in the knee joints of plg^(−/−) mice afterthe antibiotic treatment.

Example 3

Plasminogen Deficiency Impairs Bacterial Clearance

Methods

This experiment was performed in a similar manner as Example 1, exceptfor the bacterial counts. Bacterial counts. At days 2, 3, 4, 5, 7, 14,and 28 post bacterial injection., the knee joints were taken andhomogenized in 1 ml sterile PBS. After serial dilutions, the solutionsof homogenates were spreaded on LB agar plates and incubated at 37° C.overnight. Viable bacterial colonies were then counted to evaluate thenumber of bacteria in each homogenate.

Results

We then investigated the bacterial growth, in the knee joints ofplg^(+/+) and plg^(−/−) mice after bacterial arthritis induction. Asshown in Table 1, in plg^(+/+) mice, the quantity of S. aureus in theinfected knee joints declined immediately from day 2 after bacterialinjection. At day 7, S. aureus was undetectable in 50% (7/14) of theplg^(+/+) mice. At day 14. S. aureus was undetectable in 80% (8/10) ofthe plg^(+/+) mice, and less than 1×10³ CFU was found in the other 2mice. At day 28, the bacteria were completely eliminated in allplg^(+/+) mice. In sharp contrast, all of the plg^(−/−) mice had S.aureus in the injected knee joints throughout the entire experimentalperiod (Table 1). At day 28, the amount of S. aureus in the inoculatedjoints of plg^(−/−) mice was 27-fold higher than the amount injected atday 0. These data show that clearance of bacteria in the knee joints ofplg^(−/−) mice were impaired, suggesting that plasmin is involved in thebacterial-killing process during host defense against infection.

TABLE 1 Recovery of bacteria in knee joints of plg^(+/+) and plg^(−/−)mice after intraarticular injection of 1 × 10⁶ CFU of S. aureus PhilipsDays after bacterial Mean number of bacteria* Incidence** injectionPlg^(+/+) Plg^(−/−) Plg^(+/+) Plg^(−/−) 2 (7.6 ± 3.7) × 10⁵ (2.9 ± 1.5)× 10⁶ 5/5 3/3 3 (3.7 ± 0.9) × 10⁵ (2.3 ± 0.5) × 10⁶ 5/5 5/5 4 (3.0 ±0.6) × 10⁵ (1.7 ± 0.3) × 10⁶ 5/5 5/5 5 (2.2 ± 0.5) × 10⁵ (1.2 ± 0.2) ×10⁶ 5/5 5/5 7 (8.6 ± 1.3) × 10³ (1.9 ± 0.3) × 10⁶  7/14 11/11 14 (4.0 ±2.0) × 10² (3.7 ± 0.6) × 10⁶  2/10 10/10 28 undetectable (2.7 ± 0.6) ×10⁷  0/10 10/10 *Average bacterial counts in knee joints that haddetectable levels of living bacteria. **Data represent the number ofknee joints with detectable levels of living bacteria divided by thetotal number of knee joints examined in each group.

Example 4

Infiltration of the Infected Joints By Macrophages and Neutrophils isNot Overtly Impaired in plg^(−/−) Mice

Methods

This experiment was performed in a similar manner as Example 1, exceptfor the immunohistochemical analysis and inflammatory cell counting.

Immunohistochemical analysis. Paraffin-embedded sections (8-μm) weredeparaffinized, rehydrated, and the endogenous peroxidase activity wasblocked with 0.3% H₂O₂ for 10 min. After incubation with 5% rabbit serumat room temperature for 20 minutes, the slides were incubated at 4° C.overnight with rat anti-mouse primary antibodies against macrophages(clone F4/80, MCAP497, Serotec, UK) or against neutrophils (MCA771G,Serotec, UK), respectively. Thereafter, for macrophage and neutrophilimmunostainings, the slices were rinsed and incubated Further with goatanti-rat IgG (SC-2019, Santa Cruz, Calif.) antibody at room temperaturefor 1 hour.

Inflammatory cell counting. The total number of cells per sectionderived front an area of 5 fields at ×400 magnification. The wholecounting procedure was done in duplicates for each section and a meanvalue per section was calculated. Three independent sections werecounted from each joint and five joints from separate mice of the samegenotype were used at each time point.

Results

Immunohistochemical analysis was performed to investigate theinfiltration of macrophages and neutrophils into the infected kneejoints in plg^(+/+) and plg^(−/−) mice. Inflammatory cell infiltrationwas quantified by counting the number of positively-stained cellspresenting on the sections. Three independent sections were counted fromeach joint and 5 joint samples from each genotype at each time pointwere included in the study (FIG. 3A and 3B). In both plg^(+/+) andplg^(−/−) mice, similar numbers of neutrophils and macrophages hadinfiltrated into the synovium of the infected knee joints within 24hours of bacterial injection. Thereafter at days 7 and 14, the numbersof accumulated neutrophils and macrophages in both plg^(+/+) andplg^(−/−) mice had increased significantly as compared to that of day 1.These data indicate that, from within 24 hours till day 14 afterbacterial injection, neither neutrophil nor macrophage infiltration tothe infected joints was impaired in plg mice as compared to plg^(+/+)mice. However, although the infiltration of neutrophils and macrophageswas overtly impaired in mice, the normal function of these cells,especially the ability to kill bacteria, was severely compromised.

Example 5

Systemic Supplementation of plg^(−/−) Mice with Human Plasminogen (hPlg)Restored the Normal Host Defense Against S. aureus Infection

Methods

This experiment was performed In a similar manner as Example 1, exceptfor the administration of human plasminogen to some of the mice andwestern analysis.

Supplementation of plg^(−/−) mice with human Plasminogen.

For experimental protocol 1, arthritis was induced by local inoculationof 1×10⁶ CFU of S. aureus Phillips in 10 μl sterile PBS into both kneejoints of mice. 6 hours before bacterial inoculation, each of 6plg^(−/−) mice was supplemented with 1 mg human plasminogen (hPlg)(Biopool, Umeå, Sweden) in 100 μl sterile PBS by intravenous (i.v.)injection at 24-hour intervals for 7 days. As controls. 6 plg^(+/+) and6 plg^(−/−) mice were injected with sterile PBS alone at 24-hourintervals during 7 days experimental period. Mice were sacrificed at day7 after bacterial injection and knee joint samples were dissected,decalcified and processed for histological and immunohistochemicalanalysis.

For experimental protocol 2, arthritis was induced as experimentalprotocol 1. From day 7 after bacterial inoculation, each of 6 plg^(−/−)mice was supplemented with 1 mg hPlg in 100 μl sterile PBS by i.v.injection. The same amount of plasminogen was injected thereafter at24-hour intervals for 7 days. As controls, 6 plg^(+/+) and 6 plg^(−/—)mice were injected with sterile PBS alone at 24-hour intervals for 7days. Mice were sacrificed at day 14 post injection and knee jointsamples were dissected, decalcified and processed for histologicalanalysis.

Results

To confirm that plasmin plays a role in host defense against S.aureus-induced bacterial arthritis, we investigated the development ofbacterial arthritis in plg^(−/−) nice supplemented with hPlg. We firstperformed an experiment where 6 plg^(−/−) mice were supplemented withhPlg from day 0 to day 7 after bacterial injection. We then investigatedthe effects of supplementation of hPlg on 6 plg^(−/−)mice that hadalready developed bacterial arthritis for 7 days. As shown in FIG. 4Aand 4B, when plg^(+/+) mice received sterile PBS, moderate levels ofinflammation were observed in the synovium and the bone structure wasrelatively intact. As shown in FIG. 4C and 4D, when plg^(−/−) micereceived only sterile PBS, necrotic tissue was observed in parts of thesynovial tissues, the inflammation and tissue destruction were much moresevere than in the plg^(+/+) group. When plg^(−/−) mice weresupplemented with hPlg from day 0 to day 7 (FIG. 4E), thehistopathological features resembled that in plg^(+/+) mice receivingPBS. As shown in FIG. 4F, when plg^(−/−) mice were given hPlg from day 7to day 14, the joint morphology was substantially more intact than thatin plg^(−/−) mice receiving PBS, and the levels of inflammation werecomparable to the plg^(+/+) control group. In addition, there was verysmall area necrotic tissue in the synovium in the group receiving. hPlg.As shown in Table 2, the ability to kill bacteria was also restored whenplg^(−/−) mice were supplemented with hPlg for 7 days after bacterialinjection. These data clearly show that plasmin(ogen) is essentiallyrequired for the clearance of S. aureus in arthritic knee joints and forthe integrity of host defense against infection.

TABLE 2 Recovery of bacteria in the infected knee joints aftersupplementation of plg^(−/−) mice with plasminogen Mean bacterial countsin the joints* (CFU) Plg^(+/+) mice Plg^(−/−) mice Plg^(−/−) mice givengiven PBS given PBS human plasminogen Days 0-7 (3.2 ± 2.0) × (1.3 ±0.12) × 10⁶, n = 6 (1.3 ± 0.43) × 10³, n = 6 10³**, n = 6 Days 7-14 (7.2± 4.3) × (2.2 ± 0.59) × 10⁶, n = 6 (1.8 ± 0.56) × 10², n = 6 10³**, n =6 *Average bacterial counts in knee joints that had detectable amountsof living bacteria. **P < 0.01. The group of plg^(−/−) mice given humanplasminogen was compared with the group of plg^(−/−) mice given PBS.

Example 6

Local Supplementation of plg^(−/−) Mice with Human Plasminogen (hPlg)Restored the Normal Host Defense against S. aureus Infection

Methods

Bacterial arthritis was induced by local inoculation of 1×10⁶ CFU of S.aureus Phillips in 10 μl sterile PBS into both knee joints of mice. 15minutes after bacterial inoculation, one side of the knee joints of 6plg^(−/−) mice was supplemented with 40 μl of human plasminogen (10μg/μl in PBS. Biopool, Umeå, Sweden) by local injections around the kneejoint tissue. Thereafter human plasminogen was supplemented at 24-hourintervals for 7 days. As controls for local injections, 6 plg^(−/−) micewere locally injected around the knee joint tissue with 40 ul of sterilePBS alone at 15 minutes after bacterial inoculation, and thereafter at24-hour intervals during 7 days experimental period. As controls forwild-type in ice, 2 plg+/+ mice were given 40 ul of sterile PBS alone at15 minutes after bacterial inoculation, and thereafter every 24 hoursfur 7 days. As controls for plg−/− mice with systemic injections, 2plg−/− mice were given 100 μl human plasminogen (10 μg/μl) intravenously1 hour before bacterial inoculation and thereafter every 24 hours for 7days.

Mice were sacrificed at day 7 after bacterial inoculation and the kneejoints were taken and homogenized in 1 ml sterile PBS. After serialdilutions, the solutions of homogenates were spreaded on LB agar platesand incubated at 37° C. overnight. Viable bacterial colonies were thencounted to evaluate the number of S. aureus bacteria in each homogenate.

Results

7 days of local injection of plasminogen to plg−/− mice inoculated withS. aureus successfully and significantly decreased the amounts ofbacteria to 100-folds as compared to the PBS local treatment in thesemice (Table 3 and FIG. 9). Both plg−/− mice with systemic injection ofhuman plasminogen or plg+/+ mice with local injection of PBS have alsosuccessfully killed S. aureus in their knee joints. These data clearlydemonstrate that local injection of human plasminogen can restore thenormal bacterial killing capacity in the plg−/− mice.

TABLE 3 Bacterial number in plg−/− and plg+/+ mice with different localand systemic treatments at day 3 after inoculation of 1 × 10⁶ CFU of S.aureus Phillips Mean number of bacteria (Mean ± SD, × Groups Number ofsamples 10⁶ CFU) Plg−/− with local 6  0.019 ± 0.044* injection of hPlgPlg−/− with local 6 1.09 ± 0.55 injection of PBS Plg−/− with systemic 20.00075 ± 0.0011* injection of hPlg Plg+/+ with local 2  0.00065 ±0.00092* injection of PBS *P < 0.05, compared to the group of plg−/−mice with local injection of PBS.

Example 7

Local Supplementation of plg^(+/+) Mice with Human Plasminogen Enhancesthe Host Defense Against S. aureus Infection

Methods

Bacterial arthritis was induced by local inoculation of 1×10⁶ CFU of S.aureus Phillips in 10 μl sterile PBS into knee joints of mice. 15minutes after bacterial inoculation, one side of the knee joints of 7plg^(+/+) mice was supplemented with 50 μl of human plasminogen (hPlg,10 μg/μl in PBS, Biopool, Umeå, Sweden) by local injections under theknee skin and around the knee joint tissue. Thereafter human plasminogenwas supplemented in the same pattern at 24-hour intervals from day 0 today 2. As controls for local injections, 7 plg^(+/+) mice were locallyinjected under the knee skin and around the knee joint tissue with 50 ulof sterile PBS alone at 15 minutes after bacterial inoculation, andthereafter the same local injections were performed at 24-hour intervalsfrom day 0 to day 2 of the experimental period.

Mice were sacrificed at day 3 after bacterial inoculation and the kneejoints were taken and homogenized in 1 ml sterile PBS. After serialdilutions, the solutions of homogenates were spread on LB agar platesand incubated at 37° C. overnight. Viable bacterial colonies were thencounted to evaluate the number of S. aureus bacteria in each homogenate.

Results

Local injection at the knee joints of human plasminogen for 3 days inplg+/+ mice successfully and significantly reduced the living S aureusnumber for 5 folds as the the control plg+/+ group treated PBS. Thesedata clearly demonstrate that human plasminogen is a potentpro-inflammatory factor that potentiates the host defense againstbacterial infection even in wild-type animal. These data (Table 4, FIG.10) further indicate that plasminogen is a novel anti-infectious drugcandidate for clinical use.

TABLE 4 Bacterial number in wild-type (plg+/+) mice locally injectedwith human plasminogen or PBS at day 3 after inoculation of 1 × 10⁶ CFUof S. aureus Phillips Number of Mean number of bacteria Groups samples(Mean ± SE, × 10⁶ CFU) Plg+/+ with local injection of hPlg 7 0.031 ±0.011* Plg+/+ with local injection of PBS 7 0.14 ± 0.047 *P < 0.05, ascompared to the group of Plg+/+ mice with local injection of PBS.

Example 8

Supplementation of plg^(−/−) Mice with Plasminogen Increased the IL-6Protein Expression in the Infected Knee Joints

Methods

This experiment was performed in a similar manner as Example 5, exceptfor the immunohistochemical staining of IL-6.

For IL-6 immunostaining, the slices were rinsed and incubated with Swineanti-rabbit IgG antibody (P0217, DAKO, Denmark) at room temperature for1 hour. The chromogenic reaction was developed by DAKO substrate kit(K3464, DakoCytomation AEC substrate, USA) and the slides werecounterstained with hematoxylin. Slides incubated with rabbit seruminstead of the primary antibody served as negative control, they allshowed negative.

Results

IL-6 has been reported to be involved in lymphocyte activation, growthand differentiation and lack of IL-6 enhances the susceptibility toinfection. We therefore investigated whether plasmin has any effect onIL-6 expression during bacterial arthritis. Samples for tissue sectionwere taken during supplementation of plg^(−/−) mice with hPlg andperformed by immunohistochemical stainings. When plg^(−/−) mice receivedsterile PBS for 7 days after bacterial injection, the IL-6 proteinlevels were significantly lower as compared to plg^(+/+) mice (FIGS. 5Aand 5B). As shown in FIG. 5C, when plg^(−/−) mice received hPlg for 7days after bacterial injection, the IL-6 protein expression wasincreased to a similar level as the plg^(−/−) mice. These data show thatplasmin is involved in the regulation of IL-6 expression in knee jointsduring bacterial arthritis.

Example 9

Higher Levels of IL-10 Expression in plg^(+/+) Joints as Compared toplg^(−/−) Joints

Methods

This experiment was performed in a similar manner as Example 1, exceptfor the western blots analysis.

Western Blots Analysis.

At days 3 and 7 after bacterial injection, mice were sacrificed and thewhole knee joints were collected. Joints were homogenized and lysed inNP-40 buffer (0.5% Nonidet P-40, 50 mM Tris-HCl (pH 7.4), 150 m.114NaCl, 1 mM NaF, 1 mM EDTA. 1 mM Na₃VO₄, 0.25 mM PMSF, 5 μ/ml aprotinin,1 μg/ml leupeptin, 1 μg/ml pepstatin, and 15% glycerol) for 30 min onice. The lysates were adjusted for equal protein concentration. Thewestern blot analysis were performed as described (12), using agoat-anti-mouse IL-10 antibody (AF-417-NA, R & D systems, UK) and mousemonoclonal antibody against β-actin (Sigma-Aldrich Sweden AB, Stockholm,Sweden). Anti-goat and anti-mouse secondary antibodies conjugated withhorseradish peroxidase (HRP) were from Biorad (Hecules, Calif., USA).

Results

Previous studies have demonstrate that interleukin-10 (IL-10) haveanti-inflammatory effects in animal model of septic arthritis (13). Tostudy whether plasmin has any effects on IL-10 expression duringbacterial arthritis, western blot analysis was performed to compare theIL-10 levels between plg^(+/+) and plg^(−/−) mice. At days 3 and 7 afterbacterial injection, joint homogenates were lysed and performed withwestern blotting for IL-10. Plg^(−/−) mice have dramatically lower IL-10levels compared with plg^(+/+) mice in un-infected, knee joints. (FIG.6A, lanes 1 and. 2, respectively). At day 3, the IL-10 levels in bothgenotype mice were elevated, although plg^(−/−) still have markedlylower levels of IL-10 as compared to plg^(+/+) mice. (FIG. 6A, lanes 3and 4). At day 7, plg^(−/−) mice still have same levels of IL-10 ascompared to day 3, whereas the IL-10 levels were decreased in plg^(+/+)mice (FIG. 6A, lane 5, lane 6). Taken together these data suggest thatIL-10 in concert with IL-6 may regulate inflammatory process duringbacterial arthritis and plasmin is involved in the regulation of IL-6and IL-10 expression.

Example 10

uPA is Important for Host Defense and Tissue Remodeling Against S.aureus Induced Knee Infection

Methods

At day 0, bacterial arthritis was induced by local intra-articularinoculation of 1×10⁶ CPU of S. aureus Phillips in 10 μl sterile PBS intoknee joints or wild-type and uPA-deficient (uPA−/−) mice (14). Mice weresacrificed on days 7, 14, 21 and 28, and the knee joints were taken andhomogenized in 1 ml sterile PBS. After serial dilutions, the solutionsof homogenates were spread on LB agar plates and incubated at 37° C.overnight. Viable bacterial colonies were then counted to evaluate thenumber of S. aureus bacteria in each homogenate.

In another experiment, uPA-deficient and wild-type mice were inducedwith bacterial arthritis by local intra-articular inoculation of 1×10⁶CFU of S. aureus Phillips in 10 μl sterile PBS into knee joints. At days7, 14, and 28 after bacterial injection, mice were sacrificed andsamples of whole knee joints were collected for histological analysis.In brier, knee joints were first fixed in 4% paraformaldehyde, embeddedin paraffin, and thereafter 8-μm sections were prepared. Slidescontaining tissue sections were stained with Safranin-O for histologicalanalysis. At least 10 knee joints were included in each experimentalgroup.

Results

After induction of S. aureus-induced bacterial arthritis in wild-typeand uPA-deficient mice. In the wild-type group, the mean bacterialnumber is continuously decreasing from day 7 to day 28. At day 14, 4 outof 7 mice have cleaned bacteria completely. However, in uPA-deficientgroup, the mean bacterial number is basically constant from day 0 to day28 (FIG. 11). Although bacterial number were decreasing after day 14,there was no significant different between day 14 and day 28. Althoughthe experiment was terminated at day 28 after bacterial inoculation, itis highly unlikely that uPA-deficient mice are able to kill the bacteriaat later time points because the knee joint has totally demolished afterday 14. From day 14 and on extensive levels of necrotic tissue areaccumulated in the uPA-deficient knee joints. For histologicalexaminations, whereas wild-type mice showed transient inflammation atday 7 and the levels of inflammation quickly subsided thereafter,uPA-deficient mice showed persistent tissue inflammation and edema,extensive tissue destruction and formation of necrotic tissue throughoutthe experimental period (FIG. 12). These data (Table 5, FIGS. 11 and 12)clearly demonstrate that uPA is important in bacterial killing duringthe host, defense against S. aureus-induced arthritis, and furtherimplicate that the components of the plasminogen activator pathway playscritical roles during host defense against infection.

TABLE 5 Incidence and bacterial number in uPA-deficient and wild-typemice after induction of S. aureus-induced bacterial arthritis.uPA-deficient Wild-type Bacterial number Bacterial number Days (CFU)Incidence* (CFU) Incidence* P value** Day 0 1.0 × 10⁶ 2/3 1.0 × 10⁶ 2/3Day 7 5.6 × 10⁵ ± 1.7 × 10⁵ 6/6 1.1 × 10⁴ ± 7.9 × 10³ 6/6 0.0103 Day 148.8 × 10⁵ ± 2.6 × 10⁵ 7/7 7.5 × 10² ± 4.2 × 10² 3/7 0.006 Day 21 3.7 ×10⁵ ± 8.4 × 10⁴ 5/5 6.0 × 10² ± 6.0 × 10² 1/5 0.0023 Day 28 2.8 × 10⁵ ±4.5 × 10⁴ 5/5 6.0 × 10² ± 6.0 × 10² 1/5 0.0003 *incidence is defined asthe proportion of the number of infected knee joints to the number oftotal knee joints examined at that time point. **P value is calculatedby the comparison of bacterial numbers of all the knee joints betweenuPA-deficient and wild-type mice at respective time point.

Example 11

Higher Levels of Bodyweight Loss and Severity of Arthritis in BacterialArthritis of plg−/− Mice After Intravenous Injection of 1×10⁶ CFU of S.aureus Phillips in 200 μl Sterile PBS

Methods

Bacterial arthritis was induced in plg+/+ and plg−/− mice by intravenous(i.v.) injection of 1×10⁶ CFU of S. aureus Phillips in 200 μl sterilePBS. Mice were followed up individually every day after inoculation.Paws were inspected every 24 hours, and all mice were sacrificed at day21 after inoculation. Arthritis was defined as visible joint swellingand/or erythema of palm, wrist and ankle. To evaluate the intensity ofarthritis, a clinical scoring (arthritic index) was carried out, using asystem where macroscopic inspection, yielded a score of 0-5 points foreach paw. (0=normal, 1=marginal swelling or erythema; 2=mild swellingand erythema; 3=moderate swelling and erythema; 4=marked swelling anderythema; 5=marked swelling and deformity). The total score wascalculated by adding the scores from all 4 paws for each animal tested,resulting in an arthritic score ranging from 0 to 20 for each individualmouse. The weight of the mice was determined each day from day 0 to day21.

Results

To evaluate whether plasminogen deficiency affects the bacterialinvasion to the joints in the intravenous injection induced bacterialarthritis model, the onset day and incidence of arthritis, defined asmarginal swelling and erythema, were followed (Table 6) every 24 hoursafter bacterial inoculation. The onset day in plg^(+/+) and plg^(−/−)mice was 5.0±2.2 and 4.4±2.0 respectively (P=0.5021). Furthermore, theincidence of arthritis between plg^(+/+) and plg^(−/−) mice wereidentical. These results indicate that both plg^(+/+) and plg^(−/−) miceare susceptible to S. aureus induced arthritis by intravenousinoculation. However, a striking observation was noticed in this study,38% (12/32) of the plg^(−/−) mice were paralysed in the hind part,whereas only 3.3% (1/30) of plg^(+/+) mice showed paralysis.

In order to follow the general health situation after intravenousinoculation of S. aureus, the weight of each mouse was measured everyday during the experimental period. As shown in FIG. 13, during thefirst week of infection, both and plg^(+/+) and plg^(−/−) mice showed asubstantial weight loss, which reached the maximum 24% and 26% of bodyweight at clay 7, respectively, 7 days after infection, the body weightof plg^(+/+) mice had gradually increased. In contrast, significantweight loss was continuously observed in plg^(−/−) mice throughout theexperiment (p<0.05).

The clinical development of bacterial arthritis was also followed for 3weeks in plg^(+/+) and plg^(−/−) mice after inoculated i.v. with 1×10⁶S. aureus. As shown in FIG. 14, both plg^(+/+) and plg^(−/−) micedeveloped bacterial arthritis. There was no difference in the severityof inflammation during the first 3 days between and plg^(+/+) andplg^(−/−) mice. However, from day 7 and on, the plg^(−/−) mice had moresevere inflammation than plg^(+/+) mice, and the difference wassignificant. (p0.05). In plg^(+/+) mice, the severity reached peak atday 14 and gradually subsided thereafter. At the end of the experiment,8 out of 28 plg^(+/+) mice recovered from the arthritis. In plg^(−/−)mice, the severity of arthritis increased during the whole experiment,and none of the plg^(−/−) mice recovered from the arthritis at the endof the experiment. Taken together, these data clearly indicate thatplasminogen is not essential in the invasion of bacteria, from bloodcirculation, but essential in host defense against bacterial infectionin the knee joints and in maintaining the health situation duringbacterial arthritis.

TABLE 6 Comparison of the main features bacterial arthritis in plg+/+and plg−/− mice after intravenous injection of 1 × 10⁶ CFU of S. aureusPhillips in 200 μl sterile PBS Plg^(+/+) Plg^(−/−) Onset day (Mean ±SEM) 5.0 ± 2.2 4.4 ± 2.0 Incidence of bacterial 93% (28/30) 93% (30/32)arthritis Incidence of paralysis 3.3% 37.5% Incidence of necrosis in 0 100% infected joint Body weight at day 21 22.8 g 16.7 g

Example 12

More Severe Development of Tissue Destruction and Formation of NecroticTissue in Bacterial Arthritis of plg−/− Mice After Intravenous Injectionof 1×10⁶ CFU of S. aureus Phillips in Sterile PBS

Methods

Bacterial arthritis was induced in plg+/+ and plg−/− mice by intravenous(i.v.) injection of 1×10⁶ CFU of S. aureus Phillips in 200 μl sterilePBS. Mice were followed up individually every day after inoculation.

To perform histopathological examinations of the ankles and paws, anklesand paws were dissected and fixed in 4% buffered formalin for 24 hours.Fixed tissues were decalcified for 3 weeks in 15% EDTA, dehydrated, andembedded in paraffin. 8 μm sections of the wrist-joints were stainedwith Safranin-O and counterstained with fast green/iron hematoxylin.

For immunohistochemical analysis, Saraffin-embedded sections (8 μm) weredeparaffinized and rehydrated in ethanol and distilled water. Endogenousperoxidase activity was blocked with 3% H₂O₂ for 10 minutes. Thenincubate for 20 mM at room temperature with 5% rabbit serum for fibrindetection. Slides were then overlaid with Goat anti-mouse fbn/fbg atroom temperature for 30 min. After washing, overlaid rabbit IgGanti-goat IgG for 20 min at RT. After washing, overlaid PAP for 20 minat RT. The color was developed by (DAKO Substrate chromogen system AEC)Kit, and after washing counterstained with Mayer's hematoxylin.

Results

To determine whether the observed persistence of joint inflammation wasassociated with histological changes, paws joints from infected andplg^(+/+) and plg^(−/−) mice were performed histological analysis. Thejoints were stained with safranin-O, and counterstained with fastgreen/iron and hematoxylin. As shown in FIG. 15, in plg^(+/+) mice, oneday alter onset day, the inflammation was very slight (FIG. 15A). 3 daysafter onset day, the synovial membrane becomes hyperplastic but thecartilage was intact (FIG. 15B). 7 days after onset day, inflammatorycells had infiltrated into the joint cavity (FIG. 15C). 14 days afteronset day, the synovial membrane was much thicker than that in day 7(FIG. 15D). In plg^(−/−) mice, as shown in FIG. 15E, one day after onsetday, the inflammation level was also slight. However, 3 days after onsetday, the synovial membrane was much thicker than that on day 1, and theinflammatory cells started to invade the bone (FIG. 15F). 7 days afteronset day, the inflammatory cells filled with the whole paw joint, andsome parts of bone had degraded (FIG. 15G). 14 days after onset day,most bones and cartilage was completed degraded (FIG. 15H). In addition,14 days after onset day, in plg^(+/+) mice, although the inflammationwas severe, the severity was very slight compare to plg^(−/−) mice. Theclinical findings were verified by the histopathological examinationwhich showed clearly that the plg^(−/−) mice displayed a significantlyhigher level of both cartilage and bone destruction. Together, theplg^(+/+) mice exhibited a significantly less severe arthritis thanplg^(−/−) mice. The plasmin has a beneficial effect on the tissueremodeling.

To examine the development and resolution of the arthritis at thecellular level, microscopic analyses for necrotic tissue were performedin plg^(−/−) and plg^(+/+) mice. Necrotic tissue in the joint was foundas early as day 3 after bacterial inoculation in plg^(−/−) mice. Asshown in FIG. 16 and FIG. 15G,H, large area of necrotic tissue was foundin plg^(−/−) mice. In contrast, little necrosis was found in plg^(+/+)mice, even, they have severe inflammation. These data indicate that plgdeficient mice unable to remove necrotic tissue and cause tissuedestruction.

Based on the established role of plasminogen activation in fibrinolysis,loss of plasminogen results in increased fibrin deposition. Fibrincontent in knee joints was analyzed by fibrin immunohistochemistry (FIG.17). However, we found similar levels of fibrin deposition in allswollen joints of the plg^(+/+) control mice as of plg^(−/−) mice. Thesedata clearly indicate that fibrin deposition probably is not the reasonfor the more severe bacterial arthritis observed in Plg−/− mice.

Example 13

Plasminogen is Important During Host Defense Against S. aureus-InducedInfection During the Healing of Incisional Wounds in plg−/− Mice

Methods

To induce incisional wound, first the dorsal sides of plg−/− mice werecarefully shaved using a hair clipper and cleaned using 70% ethanol.Thereatler, a 15 mm long incision was induced along the midline on thedorsal side of the mouse. 15 min later, 1×10⁷ CFU of S. aureus in 10 ulof PBS was topically applied and spreaded onto the open wounds.Furthermore, 50 ul of plasminogen was injected subcutaneously at twosites of the two sides of the wound openings, 5 mm away from the woundopening. For control plg−/− mice, only 50 ul of PBS was locally injectedto the open wound inoculated with bacteria. Thereafter, 50 ul ofplasminogen (10 ug/ul) or PBS were injected every 24 hours at similarfashion as performed at day 0 until day 10. At day 11, mice were killedand wound samples, around the wound borders and below tissue, werecarefully dissected out and homogenized in 1 ml sterile PBS. Afterserial dilutions, the solutions of homogenates were spreaded on LB agarplates and incubated at 37° C. overnight. Viable bacterial colonies werethen counted to evaluate the number of S. aureus bacteria in eachhomogenate.

Results

In order to investigate if plasminogen plays similar bacterial killingfunctions in an open wound infection model as in bacterial arthritismodel, plg−/− mice were induced with incisional wounds and furtherinoculated locally with 10⁷ CFU of S. aureus Phillips. Thereafter thesemice were either locally injected with human plasminogen or control. PBSfor 10 days. Bacterial recovery from the tissue samples of these miceshow that local treatment of human plasminogen successfully lowered thenumber of bacteria for 10 folds as compared to the control PBS treatedplg−/− mice (Table 7, FIG. 18). These data clearly show that plasminogenplays a critical role in host defense (e.g. killing of bacteria) againstinfections accompanied with open wound. Furthermore, local injection orplasminogen also greatly improved the healing of the infected wounds(FIG. 19). Altogether, these results indicate that plasminogen isessential in host defense against different types of traumas, asevaluated by the host defense against, two types of bacterial arthritis,healing of open wounds and host defense against open wound infection.

TABLE 7 Bacterial number in plg−/− mice with local treatments of hPlg orPBS at day 11 after incisional wounding inoculated with 1 × 10⁷ CFU ofS. aureus Phillips Mean number of bacteria per gram tissue Number of(Mean ± SE, × Groups samples 10⁵ CFU/g) Plg−/− with local injection ofhPlg 4  2.6 ± 2.5* Plg−/− with local injection of PBS 2 26.8 ± 25.2 *P <0.05, compared to the group of plg−/− mice with local injection of PBS.

Example 14

Plasminogen is Important in Host Defense Against S. aureus-InducedInfection During the Healing of Burn Wounds in plg−/− Mice

Methods

To induce the scald burn model, mice are first put asleep by anesthesia.Thereafter the to-be-burned area is carefully shaved and placedvertically and freely by a 25 g 100 degree hot metal bar with the helpof forceps for 6 seconds. The metal bar is pre-heated in hot water atboiling temperature. Six seconds of burn induces severe thermal injuryto the area. Once the area is scald burned, the surface of mouse back iscarefully wiped off to get, rid of excessive water. Around 15 min later,30 ul of 1×10⁶ CFU of S. aureus is injected just subcutaneously at thecenter of the burned area. Another 15 min later, 50 ul of plasminogen(10 ug/ul) is injected subcutaneously into two sites around the edge ofthe scald burned area, 25 ul per site. For control plg−/− PBS group, 50ul of PBS was injected in the same fashion as plasminogen. Thereafterfrom day 0 to day 9, daily injection is performed either above-below orleft-right at the burned area, with the switch every day. For plg+/+group, mice are only burned but left without any local treatment. At theend of the experiment, the wound appearance of the burns are documentedby camera and tissue samples (the burned area and beneath shallow layerof tissue) are carefully dissected out and homogenized in 1 ml sterilePBS. After serial dilutions, the solutions of homogenates were spreadedon LB agar plates and incubated at 37° C. overnight. Viable bacterialcolonies were then counted to evaluate the number of S. aureus bacteriain each homogenate.

Results

In order to investigate if plasminogen plays similar bacterial killingfunctions in a burn wound infection model as in bacterial arthritismodel, plg−/− mice were induced with burn wounds and further inoculatedlocally with 1×10⁶ CFU of S. aureus Phillips. Thereafter these mice wereeither locally injected with human plasminogen or control PBS for 9days. Bacterial recovery from the tissue samples of these mice taken atday 10 after burn show that local treatment of human plasminogensuccessfully lowered the number of bacteria for 10 folds in the plg−/−mice as compared to the control PBS treated plg−/− mice (Table 8, FIG.20), which is even lower than the bacterial number in plg+/+ micewithout local injection. These data clearly show that plasminogen playsa critical role in host defense (e.g. killing of bacteria) againstinfections accompanied with open burn wound. Altogether, these resultsindicate that plasminogen is essential in host defense against differenttypes of traumas, as evaluated by the host defense against two types ofbacterial arthritis, healing of open wounds (burn, incision) and hostdefense against open wound infection.

TABLE 8 Bacterial number in plg−/− mice with local treatments of hPlg orPBS at day 10 after burn wounds inoculated with 1 × 10⁷ CFU of S. aureusPhillips Mean number of bacteria per gram tissue Number of (Mean ± SE, ×Groups samples 10⁶ CFU/g) Plg−/− with local injection of hPlg 5  0.43 ±0.13* Plg−/− with local injection of PBS 5 4.6 ± 1.4 Plg+/+ withoutlocal injection 4 3.6 ± 2.1 *P < 0.05, compared to the group of plg−/−mice with local injection of PBS.

Example 15

Incidence of Spontaneous Chronic Otitis Media in Wild-Type andplg-Deficient Mice Methods

Experimental procedure. During an 18-week period, the mice wereanaesthetized at different time intervals by an intraperitonealinjection of a 100 μl mixture of 25 μl Dormicum® (Roche A B, Stockholm,Sweden), 25 μl Hypnorm™ (Janssen Pharmaceutica, Beerse, Belgium) and 50μl sterile water. The gross appearance of the tympanic membrane (TM)[SPELL, OUT] was carefully examined and documented under anotomicroscope. At the end of the ⁻18-week period, all animals werekilled and from 18 animals (wild-type, n=7; plg-deficient, n=11) earswere randomly divided into three groups, aimed for bacteriologicalidentification (wild-type, n=6; plg-deficient, n=6), plastic embedding(wild-type, n=4; plg-deficient, n=6) and paraffin embedding (wild-type,n=4; plg-deficient, n=10), respectively.

Plastic and paraffin embeddings. The skulk were collected for plasticand paraffin embedding as described previously (15). For morphologyplastic-embedded samples were cross-sectioned (1 μm) through the entireMEC and stained with toluidine blue. The paraffin-embedded samples werecross-sectioned (5 μm) through the entire MEC for immunohistochemistry.

Results

To study the development of chronic otitis media, 6-week old wild-typeand plg-deficient mice were selected. The status of the TMs and MECs inthe experimental mice was examined at the start of the experiment and atthe ages of 9 weeks, 13 weeks, 18 weeks and 24 weeks.Otomicroscopically, spontaneous chronic otitis media was defined as anopaque, whitish and thickened TM, with or without effusion material inthe MEC. As shown in Table 9, none of the wild-type mice (both males andfemales) developed any middle ear effusions or drainage from theexternal ear canal (EEC) during the 18-week experimental period Asrevealed by otomicroscope, their TMs were thin, transparent and normallypositioned (data not shown). In contrast, in the plg-deficient mice thenumber of ears with spontaneous otitis media gradually increased duringthe experimental period to a similar extent in both males and females.At the end of the experiment, spontaneous otitis media with variousdegrees of inflammatory changes had developed in all the ears of theremaining plg-deficient mice (Table 9).

TABLE 9 Incidence of spontaneous development of chronic otitis media inwild-type and plg-deficient mice at different ages Incidence ofspontaneous development of chronic otitis media^(a) at ages of: 13 24Mouse group 6 weeks 9 weeks weeks 18 weeks weeks Wild-type, male 0/180/18 0/16 0/16  0/16 Wild-type, female 0/18 0/18 0/18 0/18  0/18Plg-deficient, male 0/20 3/20 5/18 8/18  6/10^(b) Plg-deficient, female0/24 1/24 3/24 8/22 11/16^(b) ^(a)Data are shown as the number ofinfected/inflamed ears divided by the total number of ears in eachgroup. ^(b)When analyzed morphologically, the middle ears inplg-deficient mice that were shown normal under otomicroscope were foundto have inflammatory changes but not so pronounced, with only a thinlayer of an amorphous tissue mass in the MEC adhering to the TM.

Example 16

Identification of Bacteria Isolated from the MECs of Wild-Type andplg-Deficient Mice Methods

This experiment was performed in a similar manner as Example 8, exceptfor the bacteriological identification.

Bacteriological identification. The tympanic bullas from wild-type andplg-deficient groups were dissected free from soft tissue and a smallpiece of the bony floor of the bulla was removed with a knife. A sterileswab was dipped into the middle ear cavity (MEC) and by use of the swabthe material was further spread over Luria-broth (LB) plates andimmediately incubated at 37° C. for 48 hours. The colonies obtained wereidentified according to Cowan & Steel (16).

Results

At the end of the experiment, tympanic bulbs from 6 wild-type and 6plg-deficient mouse ears were randomly collected for bacterialidentification. As shown in Table. 10, bacteria were only found in 1 outof 6 of the wild-type samples. The bacteria were identified asStreptococcus sanguinis. However, in 5 out of 6 MEC samples obtainedfrom the plg-deficient mice bacteria were isolated. The speciesidentified were Staphylococcus aureus, Micrococcus luteus, Streptococcussobrinus and Streptococcus mutans. All of the identified bacteria wereGram-positive.

TABLE 10 Recovery of bacteria from the MECs in wild-type andplg-deficient mice at the age of 24 weeks. Mouse ear Bacterial findingsin number Wild-type Plg-deficient 1 Streptococcus sanguinisStaphylococcus aureus 2 ND^(a) Micrococcus luteus 3 ND Streptococcussobrinus 4 ND Streptococcus sobrinus and Streptococcus mutans 5 NDStreptococcus mutans 6 ND ND ^(a)ND, not detectable.

Example 17

Light Microscopical Studies of the Middle Ears in Wild-Type andplg-Deficient Mice Methods

This experiment was performed in a similar manner as Example 8, exceptfor the immunohistochemical stainings.

Immunohistochemical stainings. The paraffin-embedded sections werere-hydrated in a series of decreasing ethanol concentrations and rinsedin distilled water. Endogenous peroxidase activity was blocked with 3%H₂O₂ for 10 min and the slides were further washed in PBS. Theconsecutive sections were then treated with antibodies indicated below.In all immunohistochemical stainings, adjacent slides incubated withsera from non-immunized animals instead of the primary antibody wereused as negative controls.

For detection of inflammatory cells, rat anti-mouse monoclonal primaryantibodies against T cells (Clone 53-7.3, dilution 1:50; BD BiosciencesPharmingen, Stockholm, Sweden), B cells (Clone RA3-6B2, dilution 1:250;BD Biosciences Pharmingen), macrophages (MCAP497, dilution 1:500;Serotec, Oxford, U.K.) and neutrophils (CL8993AP, dilution 1:200:Cedarlane Laboratories, Hornby, Ontario, Canada) were used. To performimmunohistochemical stainings, slides were first retrieved and incubatedwith normal rabbit serum (Dako Patts, Copenhagen, Denmark) beforeincubated with different primary antibodies at the appropriateconcentrations. Thereafter the slides were incubated with biotinylatedrabbit anti-rat IgG (Dako Patts) and further treated using theavidin-biotin-peroxidase complex (ABC) method (Vector Laboratories,Burlingame, Calif.).

Cytokeratin was detected immunohistochemically by the peroxidaseanti-peroxidase (PAP) method using a rabbit anti-human polyclonalantibody (10550, ICN Pharmaceuticals, Aurora, Ohio) as the primaryantibody. In brief, the slides containing tissue sections were firstretrieved with 0.1% trypsin (pH 7.8) at 37° C.′ for 8 min, blocked with5% non-immunized swine serum (Dako Pans), and incubated with the primaryantibody diluted 1:100 in PBS. After this a swine anti-rabbit linkantibody (Dako Pans) was applied, followed, by a rabbit PAP complex(Dako Patts).

Fibrin(ogen) was detected immunohistochemically by the PAP method usinga goat anti-mouse polyclonal antibody (Nordic ImmunologicalLaboratories, Tilburg, The Netherlands) as the primary antibody. Afterinitial incubation with rabbit serum (Dako Patts) and then with theprimary antibody at a dilution of 1:500 in PBS, the slides wereincubated with a rabbit anti-goat link antibody (Dako Patts).Thereafter, the slides were incubated with goat PAP complex (DakoPatts).

All the slides were visualized as the brown precipitates by adiaminobenzidine (DAB) reaction (Vector Laboratories) andcounter-stained with Mayer's hematoxylin. The slides were examined bylight microscope under a Leica. DMLB microscope and images were recordeddigitally using a Leica DC 300F camera connected to a personal computer.Adjustment of contrast and brightness in individual images werepreformed using the Adobe Photoshop 7.0 software.

Results

At the end of the experiment, morphological staining was performed onplastic-embedded samples from wild-type and plg-deficient mice. As shownin FIG. 7A, the TMs and the middle ears of wild-type mice exhibited anormal structure. The TM revealed a typical thin three-layeredstructure: an outer keratinized epidermal layer, a middle lamina propriaand an inner epithelial lining contiguous with that of the MEC. Therewere no middle ear effusions detected in the MEC. However, in theplg-deficient mice, inflammatory changes were observed in all the middleears examined. The TM was thickened and adhered with an amorphous tissuemass which sometimes filled up almost the entire MEC (FIG. 7B). In manyof the samples the EEC was also filled with an amorphous tissue (FIG.7D).

At the end of the experimental period, immunohistochemical stainingswere performed to study the distribution of fibrin and keratin in themiddle ear (FIG. 7C, 7D, 7E, 7F). In wild-type mice, only weakimmunoreactivity against fibrin(ogen) and keratin was observed at theepithelial suffice of the TM (FIGS. 7C and 7E). However, in theplg-deficient mice, as shown in FIG. 7D, an amorphous tissue coveringthe mucosa of the TM and MEC was observed to have immunoreactivityagainst fibrin. The structure of the amorphous tissue varied indifferent areas and in different samples, from loose-net-like tosmear-like, or even densely packed (data not shown). Fibrinimmunoreactivity was also observed in the amorphous tissue in the EEC.In most of the plg-deficient mice, the keratin staining layer in the TMand surrounding epidermal layer of the EEC was considerably thickened(FIG. 7F).

To study the infiltration of inflammatory cells, paraffin sections fromwild-type and plg-deficient mice were stained for T cells, B cells,macrophages and neutrophils. As shown in FIGS. 8A, 8C, 8E and 8G, hardlyany inflammatory cells were detectable in the TM and in the middle earmucosa of the wild-type mice. In plg-deficient mice, however, T cells, Bcells, macrophages and neutrophils were all found in the TM and theamorphous tissue filling the middle ear and EEC (FIG. 8B, 2D, 8F and8H). T cells and B cells were relatively fewer and they were sparselydistributed in the TM and the middle ear mucosa (FIG. 8B and 8D). In themiddle ear mucosa macrophages were the most abundant inflammatory celltype (FIG. 8F) and neutrophils were the dominating cell type in theamorphous tissue extending into the EEC (FIG. 8H). Overall, theseresults suggest that plasminogen plays an essential role in protectingagainst the spontaneous development of chronic otitis media.

The foregoing Examples are presented by way of illustration and are notintended to in any way limit the scope of the present invention as setout in the appended claims. All references set out in the descriptionare incorporated by reference.

REFERENCE LIST

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1. A method for the prophylaxis, prevention and/or treatment ofinfectious disease, which comprises administering a pharmaceuticalcomposition comprising an effective amount of a compound selected fromthe group consisting of plasminogen, Lys-plasminogen, Glu-plasminogen,variants of plasminogen comprising one or more of the kringle domainsand the proteolytic domain, mini-plasminogen, and kringle domains ofplasminogen to a subject in need thereof.
 2. The method of claim 1,wherein the infectious disease is a bacterial infectious disease or aviral infectious disease.
 3. The method of claim 2, wherein thebacterial infectious disease is selected from the group consisting ofotitis media and bacterial arthritis.
 4. The method of claim 2, whereinthe bacterial infectious disease is selected from the group consistingof pneumonia, injuries in the respiratory organs caused by infections,and injuries in the joint tissues caused by infections.
 5. The method ofclaim 2, wherein the bacterial infectious disease is selected from thegroup consisting of gingivitis, periodontitis, and conjunctivitis. 6.The method of claim 1, wherein the pharmaceutical composition comprisesa combination of two or more of the compounds.
 7. The method of claim 1,wherein the pharmaceutical composition further comprises at least oneantibiotic agent.
 8. The method of claim 7, wherein the antibiotic agentis selected from the group consisting of tetracyclines, amphenicols,beta-lactams, penicillins, sulphonamides, macrolides, lincosamides,streptogamins, streptomycins, quinolones and metronidazoles.
 9. Themethod of claim 1, wherein the subject is a mammal.
 10. The method ofclaim 1, wherein the subject is deficient in plasmin or plasminogen. 11.The method of claim 10, wherein the deficiency is selected from thegroup consisting of congenital, acquired, and local.
 12. The method ofclaim 1, wherein the pharmaceutical composition is administered by amethod selected from the group consisting of systemically, locally,topically, intravenously, intramuscularly, subcutaneously, viainhalation, intrathecally, via local injection, via intra-articularinjection, and per rectally.
 13. The method of claim 1, wherein thepharmaceutical composition is administered in combination with asuitable polypeptide carrier or stabilizing agent.
 14. The method ofclaim 1, wherein the pharmaceutical composition is administered at adose of 0.05 mg to about 10 mg.
 15. The method of claim 1, wherein theadministration of the pharmaceutical composition is repeated at leastonce.
 16. The method of claim 1, wherein administering a pharmaceuticalcomposition is performed by applying a wound dressing, comprising thepharmaceutical composition, to an infected area.
 17. The method of claim1, wherein the method further comprises inducing an immune responseagainst an infectious pathogen.
 18. A pharmaceutical composition for theprophylaxis, prevention and/or treatment of infectious diseasecomprising an effective amount of a compound selected from the groupconsisting of plasminogen, Lys-plasminogen, Glu-plasminogen, variants ofplasminogen comprising one or more of the kringle domains and theproteolytic domain, mini-plasminogen, and kringle domains ofplasminogen.
 19. A kit of parts for use in the prophylaxis, preventionand/or treatment of infectious disease comprising an effective amount ofa compound selected from the group consisting of plasminogen,Lys-plasminogen, Glu-plasminogen, variants of plasminogen comprising oneor more of the kringle domains and the proteolytic domain,mini-plasminogen, and kringle domains of plasminogen and at least oneantibiotic or antimycotic agent, in separate containers.
 20. The methodof claim 9, wherein the mammal is a human.
 21. The method of claim 14,wherein the pharmaceutical composition is administered at a dose fromabout 0.5 to about 5 mg.
 22. The method of claim 15, wherein theadministration of the pharmaceutical composition is repeated at leastevery day.